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t("div",{key:o,staticClass:"event"},[t("h2",{domProps:{innerHTML:e._s(n.dates.toDateString())}}),t("h3",[e._v(" "+e._s(n.customData.method)+" ("+e._s(n.customData.who)+") ")]),t("div",{domProps:{innerHTML:e._s(n.customData.actions)}})])}))],2)},bn=[function(){var e=this,n=e.$createElement,t=e._self._c||n;return t("div",{staticClass:"legend"},[e._v("Legend: "),t("span",{staticClass:"pseudomonas"},[e._v("Pseudomonas")]),e._v(" and "),t("span",{staticClass:"burk"},[e._v("Burk")])])}],yn=(t("4de4"),t("159b"),t("5319"),t("466d"),t("d81d"),t("caad"),t("2532"),t("498a"),t("5887")),vn="Cloning: Burk \n\n28.06: Hege \n\nStreaked Escherichia coli TOP10 cells containing the plasmid pBAD. \n\n29.06: Hege \n\nPicked one single colony from the plate streaked the day before, and inoculated 3 ml LB containing Ampicillin (50 mg/ml). \n\n30.06: Hege \n\nMiniprep (QIAGEN kit) to isolate the pBAD-plasmid. \n\nUsed Nanodrop to measure the concentration of the plasmid. \nThe vector concentration was diluted to 20 ng/𝜇l for the PCR. Ran PCR to amplify the pBAD plasmid. \n\nResults: The band for the pBAD-plasmid was very weak. \nProceded with optimizing the PCR conditions. \n\n \n\n \n\nJuly \n\nWeek 27: 05.07.21-09.07.21 \n\n \n\n05.07: Hege and Jonas \n\nTroubleshooting of PCR conditions \n\nGradient PCR \n\n* Used protocol for Phusion Polymerase \n\n* Buffer: HF buffer \n\n* Results: Weak band at ∼4000 bp, unspecific products at lower temperatures. \n\n* Needs further troubleshooting. \n\nGradient PCR with GC buffer \n\n* Protocol for Phusion Polymerase \n\n* Results: Strong, single bands at 64 ℃ and 63, ℃ \n\n* The reaction mix with the two bands at 64 ℃ and 63, ℃ were mixed and treated with DpnI at 37 ℃ for 1 h to digest the methylated plasmids. \n\n \n\n06.07: Hege and Helene \n\nGel electrophoresis \n\n* The PCR products from yesterday were loaded on an agarose gel \n\n* (Use tape on the comb to make a larger well) \n\n* Loading dye (6x): 40 𝜇l sample + 6,7 𝜇l loading dye. \n\n* DNA ladder: 1,5 𝜇l \n\n* Note: Load the ladder as far away from the sample as possible \n\n* Run the gel at 90 V for 30 min \n\n \n\nGel extraction: Biolab Monarch DNA extraction kit \n\n* Take the gel to the imager and use the shield to protect against UV. \n\n* Cut out the band at the correct size with a scalpel. Cut clean cuts. \n\nNanodrop \n\n* Measure the concentration of the purified plasmid. \n\n* Result: 43 ng/ 𝜇l \n\n \n\nGibson cloning: g-block of Dehalogenase gene from Burkholderia cepacia (IDTDNA) \n\n* Used 3:1 ratio of vector to insert \n\nVolume of vector: 100 ng/43 ng/𝜇l = 2.3 𝜇l \n\nVolume of insert: 78 ng /100 ng/ 𝜇l = 0.8 𝜇l (The concentration of the vector is 100 ng/ 𝜇l) \n\nIn total: 3.1 𝜇l DNA + 3.1 𝜇l 2X Gibson mix. \n\nIncubation parameters: 50 ℃ for 15 min \n\n* Controls: Cloning with plasmid+Gibson mix, and cloning with only circular plasmid. \n\nTransformation \n\n* Transformation of Escherichia coli DH5α strain with pBAD-vector. \n\n \n\n07.07: Hege and Jonas \n\nTransformation, results: \n\n4 colonies with 3:1 ratio, 50 ℃ for 15 min. \n\n \n\nColony PCR \n\n Result: All the 4 colonies had the insert. Could then continue with miniprep and sequencing after the plates had incubated long enough to form colonies. \n\n \n\nGibson cloning: Optimalization of cloning parameters for dehalogenase(B.cepacia)+pBAD. \n\n* 6:1 ratio (double the amount of insert from last time) \n\n* Volume vector: 2.3 𝜇l \n\n* Volume insert: 1.6 𝜇l \n\n* Volume Gibson mix: 3.9 𝜇l \n\n* Incubation: 50 ℃ for 1 hour in the Thermocycler. \n\n* Transformation of DH5α with \n\nResult: No colonies. \n\n \n\n08.07: Hege and Jonas \n\nCloning refinements of dehalogenase(B.cepacia)+pBAD \n\n* Tried different ratios of insert and vector: \n\n1.3:1 ratio, 50 ℃ for 1 h: vector = 2.3 𝜇l, insert = 0.8 𝜇l \n\n2.6:1 ratio, 50 ℃ for 15 min: vector = 2.3 𝜇l, insert = 1.6 𝜇l \n\n3.1:1 ratio, 50 ℃ for 15 min: vector = 2.3 𝜇l, insert = 2.3 𝜇l \n\n* Results: \n\n3:1 ratio: 1 colony \n\n6:1 ratio: 10 colonies \n\n1:1 ratio: 23 colonies \n\n \n\nGel extraction of g-block from B.cepacia \n\n* PCR of g-block had been performed by supervisor beforehand. \n\n* Measured the concentration from Nanodrop: 110 ng/𝜇l \n\n \n\nMiniprep colonies containing vector (pBAD) + insert (g-block, dehalogenase from B.cepacia) \n\n* Followed the QIAGEN miniprep protocol. \n\n* Nanodrop of each: \n\n 88 ng/𝜇l \n\n109 ng/𝜇l \n\n195 ng/𝜇l \n\n114 ng/𝜇l \n\n \n\nSequencing \n\npBAD+dehalogenase(B.cepacia) extracted from the four colonies containing the conctruct were sent for sequencing \n\nResult: All sequences contained the correct Dehalogenase-gene. \n\n \n\nWeek 28: 12.07-16.07 \n\n \n\n12.07: Hege and Helene \n\n* PCR to amplify pBAD-BurkD vector that we sent for sequencing last week. \n\n \n\n13.07: Hege and Linda \n\n* Gel extraction of pBAD-BurkD \n\n* Result: The concentration after nanodrop was 6 ng/𝜇l. This is too low to distinguish from background. \n\n \n\nCloning: Pseudomonas \n\n \n\n14.07 and 15.07: Linda, Carina, Hege and Helene \n\n* PCR of genomic DNA from Pseudomonas aeruginosa to isolate dehalogenase gene. \n\n* Results: The size of the band is around 700 bp, and not 900 bp. Need to do further optimalizations of PCR parameters. \n\n \n\n16.07 Linda and Helene \n\nAlthough the band size was incorrect, proceeded with PCR clean up. Used the protocol from the MinElute PCR purification kit. \n\nNanodrop: 13 ng/µl. \n\n \n\n \n\nWeek 29: 19.07-23.07 \n\n \n\n20.07: Hege and Linda \n\nPCR amplification of insert PCR product \n\n* The concentration of insert was too low to send to sequencing (13 ng/𝜇l) \n\n* Used Phusion polymerase. \n\n* Diluted the sample 1:10 to get in the correct range (1pg-10ng) \n\nAnnealing temp: 61 °C \n\n \n\n* Sequencing of insert from P.aeruginosa \n\nResult: Not correct gene – ordered new primers. \n\n \n\nWeek 30: 26.07-30.07 \n\n26.07-30.07: No lab work \n\n \n\nAugust \n\n \n\nWeek 31: 02.08-06.08 \n\n \n\nProtein expression: Burk \n\n05.08: Jonas and Barathy \n\nTransformed BL21 competent cells with pBAD+Burk.D \n\nPlated and incubated over night at 37 °C \n\n06.08 Jonas and Barathy \n\nGot colonies from the transformation the day before. \n\nStored in fridge for later expression \n\n \n\nCloning: Pseudomonas \n\n06.08: Jonas and Barathy \n\nGot new primers for Dehalogenase from P.aeruginosa \n\nPCR on genomic DNA from P.aeruginosa to isolate the Dehalogenase gene. \n\nVector: pBAD+BurkD. \n\nAnnealing temp.: 68 °C \n\nStored in freezer for later use. \n\n \n\nCloning: Burk \n\n06.08: Jonas and Barathy \n\nPCR to amplify pBAD-BurkD construct \n\nAnnealing temp.: 62 °C \n\nStored in freezer for later use. \n\n \n\nWeek 32: 09.08-12.09 \n\nCloning: Pseudomonas \n\n09.08: Linda \n\nDpnI digest of insert from PCR performed 06.08 (pBAD-Pseudo) \n\nGel electrophoresis to check if the band is at the correct size (903 bp) \n\nLadder: N3232L \n\nResult: Band right below 1000 bp. \n\nGel extraction using Biolab Monarch DNA extraction kit \n\nExtracted the band right below 1000 bp. \n\nNanodrop: 147 ng/ul \n\n \n\n Cloning: Burk \n\n \n\n09.08: Linda \n\nDpnI digest of PCR product form 06.08 (pBAD-BurkD) \n\nGel electrophoresis to check if band is correct size (3978 bp) \n\nResult: Band at ~4000 bp. \n\nGel extraction using Biolab Monarch DNA extraction kit \n\nExtracted the band at ~4000 bp. \n\nNanodrop: 57 ng/ul. \n\n \n\nProtein expression: Burk \n\n10.08: Athanasios \n\nOvernight culture with BL21 transformed with pBAD-BurkD 05.08 \n\nLB+0.1 % Glucose and 100 ug/ml Ampicillin. \n\nIncubated at 37 °C, 200 rpm overnight. \n\n \n\n11.08: Linda, Carina and Jonas \n\nProtein expression continued \n\nDiluted the overnight culture 100 times in fresh LB+Amp(100 ug/ml) +0.1 % glucose. \n\nStarted expression when OD was too high. \n\nExpression induced with 0.2 % arabinose. \n\nInduced cells were spun down and the pellet stored in the freezer. \n\n \n\nWeek 33: 16.08-20.08 \n\n \n\n17.08: Hege and Carina \n\nProtein expression continued \n\nLysed the cells that were frozen 11.08. \n\nVisualized on a SDS-PAGE gel. \n\nResult: The gel looked weired, may be something wrong with the running buffer. Must also do the expression again because the OD was too high at the point of induction. \n\n \n\n18.08: Linda and Hege \n\nTransformation of BL21 with pBAD-BurkD construct. \n\n \n\nCloning: Pseudomonas \n\n \n\n18.08: Linda and Hege \n\nCloning of Dehalogenase from P. aeruginosa into pBAD vector \n\n100 ng vector, 57 ng/ul = 1.8 ul vector \n\n23 ng insert, 14.7 ng/ul = 1.6 ul insert. \n\nTotal DNA = 3.4 ul \n\n2X Gibson mix = 3.4 ul \n\nTransformation \n\nCloning assembly transformed into DH5α competent cells \n\nResult: No colonies. Need to do further troubleshooting on the cloning conditions. \n\n \n\n19.08: Jonas and Barathy \n\nGibson assembly of Dehalogenase from P.aeruginosa into pBAD vector \n\nTried 3 different ratios of insert and vector \n\n1:1, 1:3 and 1:6 \n\nIncubated at 50 °C for 15 min. \n\nTransformation \n\nCloning assembly tranformed into DH5α competent cells. \n\nPlated and incubated overnight at 37 °C. \n\nResult: No colonies. \n\n \n\nWeek 34: 23.08-27.08 \n\nProtein expression: Burk \n\n23.08: Hege and Linda \n\nStart expression protocol \n\nMake overnight culture by inoculating 5 mL LB + Amp (100ul/ml) with transformed BL21 cells containing vector+insert. \n\nIncubate at 37 °C, 200 rpm overnight. \n\n24.08: Hege and Linda \n\nContinue protein expression \n\nOvernight culture was diluted 100 times in fresh LB medium + Amp (100 ul/ml) + 0.1% glucose. \n\nCells were incubated at 37 °C, 200 rpm until OD~0.4-0.6. \n\nInduction started by adding 0.2 % arabinose to medium and left at RT overnight at shaking. \n\n25.08: Hege and Carina \n\nExpression continued \n\nOD was normalized to make sure the same amount of cells were harvested. \n\nCells lysed and run on a SDS-PAGE gel. \n\nResult: Expression not succesful. Need further troubleshooting. Don’t use glucose in medium next time. \n\nSeptember \n\nWeek 35: 30.08-03.09 \n\nCloning: Pseudomonas \n\n02.09: Helene and Barathy \n\nPCR of genomic DNA from P.aeruginosa to isolate Dehalogenase gene \n\nPhusion DNA polymerase \n\nAnnealing temp.: 68 °C \n\nPCR of pBAD-BurkD vector \n\nPhusion DNA polymerase. \n\nAnnealing temp.: 62 °C. \n\nDpnI digest of PCR products from vector and insert. \n\n \n\n03.09: Linda and Hege \n\nGel electrophoresis of the PCR product pBAD-vector \n\nBand from vector was purified with MinElute PCR purification kit. \n\nNanodrop: 131 ng/ul, \n\nGradient PCR of insert from P.aeruginosa \n\nTried both GC buffer and HF buffer \n\nTemperature gradient: 68 °C - 58 °C \n\nResult: There was no band at ~900 bp. Try to do the PCR again. \n\n \n\nWeek 36: 06.09-10.09 \n\nProtein expression: Burk \n\n08.09: Hege \n\nProtein expression of Dehalogenase from B.cepacia. \n\nMade overnight culture by inculatig 5 mL LB+Amp \n\n09.09: Hege and Helene \n\nProtein expression continued: \n\nDilute the overnight culture 1:100: Transfere 400 ul from the overnight culture into 40 mL LB+Amp. \n\nIncubate at 37 °C until OD ~0.4-0.6. \n\nInduction was started by adding arabinose to a final concentration of 0.2 % intto the culture. Keep one fraction as the non-induced control. \n\nThe samples were incubated at room temperature overnight at shaking 200 rpm. \n\n10.09: Helene and Carina \n\nProtein expression continued \n\nThe OD of the induced and non-induced overnight cultures were normalized. \n\nThe cells were lysed using B-PER (Thermo Fischer), lysozyme, DNAse, MgCl2 and PMSF was added. \n\nLoaded and run on a SDS-PAGE gel at 225 V for 40 min. \n\nResult: The bands were hardly visible. Need to up-concentrate the cells used. \n\nCloning: Pseudomonas \n\n10.09 Linda and Helene \n\nGradient PCR of insert from P.aeruginosa \n\nTemperature gradient: 72 °C - 63 °C. \n\n \n\nWeek 37: 13.09-17.09 \n\n \n\n13.09: Helene and Carina \n\nGel extraction \n\nCombined 1-4 of PCR products of P.aeruginosa insert from PCR gradient 03.09 \n\nExctracted the bands using Monarch DNA gel extraction kit. \n\nThe DNA was eluted in 6 ul. \n\n \n\n14.09 Helene and Carina \n\nGibson assembly of inser from P. aeruginosa and pBAD-vector. \n\n6 ul insert + 1 ul pBAD vector (131 ng/ul) + 7 ul 2X Gibson mix. \n\nIncubated at 50 °C for 15 min. \n\n \n\n15.09 Helene and Carina \n\nTransformation of DH5α cells with Gibson assembly from 14.09. \n\nResult: No colonies. \n\n \n\n16.09 Helene and Carina \n\nPCR amplification and purification of insert from P.aeruginosa \n\nGC buffer. \n\nAnnealing temp: 67 °C. \n\nDNA template: genomic DNA from P.aeruginosa. \n\nTreated the PCR sample with sodium acetate and cold 100 % Ethanol for purification. Left in freeer overnight to percipitate. \n\n \n\n \n\n17.09 Helene and Carina \n\nPCR amplification and purification of insert from P.aeruginosa continued \n\nPCR sample from yesterday was loaded on gel. \n\nPerformed gel extraction. \n\nNanodrop: 67 ng/ul. \n\n \n\nProtein expression: Burk \n\n13.09: Carina and Helene \n\nProtein expression of Dehalogenase from B.cepacia. \n\nLysed the cells using a lower volume of B-PER to make the samples more concentrated. Also used more cells than last time. \n\nSamples were loaded and run on a SDS-PAGE gel at 225 V for 40 min. \n\nResults: There were bands, but need to run the gel again to get better looking bands. \n\n14.09: Helene and Carina \n\nProtein expression continued \n\nRan another gel with the samples from 13.09. \n\nResult: Visible bands at correct size. \n\n \n\nWeek 38: 20.09-24.09 \n\n \n\nCloning: Pseudomonas \n\n20.09: Helene, Carina and Linda \n\nPCR amplification of pBAD-vector \n\nAnnealing temp.: 62 °C. \n\nPhusion DNA polymerase. \n\nDpnI digest of PCR product. \n\nPCR purification \n\nPCR clean up from QIAGEN. \n\nNanodrop: 327 ng/ul. \n\n21.09: Linda and Hege \n\n Gibson assembly of insert from P.aeruginosa into pBAD. \n\nTried 3 different ratios of insert and vector: 1:1, 1:3 and 1:6. \n\nIncubated at 50 °C for 1 hour. \n\nTransformation of assembly into chemically competent cells (DH5α) \n\nPlated on Ampicillin plates and left overnight at 37 °C. \n\nResult: 2 colonies for the ratio 1:3. \n\n22.09: Hege \n\nColony PCR of colonies from cloning with pseudo+pBAD \n\nPicked one colony with a loop and inoculated the PCR reaction mix. Inoculated also \n5 mL LB+Amp. \n\nGel electrophoresis: \n\nResult: Looks like colony 2 had the insert because the band was at ~900 bp, but since there were only two colonies, proceeded with sequencing for both of the colonies. \n\n \n\n23.09: Hege \n\nMiniprep of bacteria possibly containing insert from P.aeruginosa+pBAD. \n\nMiniprep was performed using the Qiagen miniprep kit. \n\nNanodrop: \n\nColony 1: 59,9 ng/ul. \n\nColony 2: 74.1 ng/ul. \n\nThe DNA was sent for sequencing: \n\nResult: Colony 1 had the vector with the correct insert. Proceed with protein expression with this construct. \n\n \n\nProtein expression: Burk \n\n23.09: Helene and Carina \n\nProtein expression (larger scale) of Dehalogenase from B.cepacia: \n\nLB + Amp50 was inoculated by single colony containing Dehalogenase from B.cepacia in pBAD vector. Incubated at 37 °C 200 rpm overnight. \n\n24.09: Helene and Carina \n\nProtein expression (larger scale) of Dehalogenase from B.cepacia (continued): \n\n1.5 mL of overnight culture was transferred to 150 ml LB + Amp50. Incubated until OD~0.6. \n\nExpression was induced by adding arabinose to a final concentration of 0.2%. 5 mL was taken as an uninduced control. \n\nThe pellet was spun down and supernatant was removed. The pellet was freezed down at –20 °C until further use. \n\nWeek 39: 27.09-01.09 \n\n29.09: Helene and Carina \n\nMade buffers for protein purification under dentatruting conditions. \n\n28.09: Helene and Carina \n\nMade buffers for protein purification under dentatruting conditions. \n\n \n\nOctober \n\nWeek 40: 04.10-08.10 \n\nProtein expression: Pseudomonas \n\n04.10: Linda and Hege \n\nTransformation of BL21 cells with insert from P.aeruginosa in pBAD vector. \n\nAdded 1.5 ul of DNA to 70 ul of BL21 cells. \n\nAfter tranformation protocol, the cells were plated and incubated at 37 °C overnight. \n\nResult: Many colonies. \n\n \n\n06.10: Hege and Linda \n\nSmall-scale protein expression \n\nOvernight culture was performed the day before. \n\nThe expression was induced by adding arabinose to the culture in a final concentration of 0.2 %. \n\nCells were lysed using B-PER (Thermo Fischer). \n\nResult: No bands on SDS-PAGE gel. May have used too low concentration of cells in the lysis process. Have to redo the expression. \n\n \n\nProtein purification: Burk \n\n05.10: Hege and Helene \n\nProtein purification of Dehalogenase from Burkholderia cepacia under denaturing conditions. \n\nSince the protein was in the insoluble fraction, the protein purification was performed under denaturing conditions, and later refolded. \n\nStartet from pellet that was frozen down after expression at 24.09. \n\nCells were lysed using denaturing lysis buffer and sonication. \n\nUsed Ni-NTA resin to purify the His-tagged protein. \n\nBSA assay \n\nDetermine protein purification using Bovine serum albumin protein standard. \n\n \n\n07.10: Helene, Barathy and Hege \n\nProtein refolding \n\nInvestigation of refolding conditions for the protein \n\nDifferent buffers with different concentrations of NaCl was made. \n\nThe protein was diluted 1:20 in the different buffer conditions. \n\nThe samples were loaded on a SDS-PAGE gel at 225 V for 40 min. \n\n07.10: Hege and Linda \n\nSilver staining \n\nThe SDS-PAGE gel with the different samples from the refolding was silver stained. \n\nDid this 3 times to improve the result. \n\nFirst result: Got som bands from two of the refolding conditions, but the positve control (denatured protein) was not visible. Had to do it again. \n\nResult 2: Stopped the staining too soon, so the bands from the refolded proteins were not visible. The positive control (denatured protein) was not visible. Had to upconcentrate the control. \n\nResult 3: Used up-concentrated positive control. There were alot of bands, indicating that the purification was not satisfactory. No bands for the refolded proteins. Have to do the purification again. \n\nWeek 41: 11.10-15.10 \n\n11.10: Hege \n\nMedium scale expression of Dehalogenase from B.cepacia \n\nArabinose added to a final concentration of 0.2 % into a bacteria culture with OD~0.4-0.6. \n\nIncubated at 37 °C for minimum 4 hours. \n\n \n\n12.10: Hege and Linda \n\nProtein purification of Dehalogenase from B.cepacia \n\nResuspended pellet from the day before. \n\nPerformed protein purification of His-tagged protein using Ni-NTA under denaturing conditions since the protein has proven to be in the insoluble fraction. \n\nLoaded the samples on a SDS-PAGE gel. \n\nRan the gel at 225 V for 40 min. \n\nResult: Managed to purify most of the contaminats. \n\n \nProtein expression: Pseudomonas \n\n13.10: Hege \n\nSmall-scale protein expression of Dehalogenase from P.aeruginosa \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n ",Fn=function(e){Object(h["a"])(t,e);var n=Object(d["a"])(t);function t(){var e;return Object(c["a"])(this,t),e=n.apply(this,arguments),e.dates=[],e.date=new Date,e}return Object(l["a"])(t,[{key:"events",get:function(){var e=this;return this.dates.filter((function(n){return n&&n.dates.toDateString()===e.date.toDateString()}))}},{key:"mounted",value:function(){var e=this,n=vn,t=[/\n[\w ]+\n/g,/\d+: \d+.\d+\d+-\d+.\d+\d+/g,/\nWeek[^\n]*/g,/\*/g],o=/(?:Cloning|Protein expression): (?:Burk|Pseudomonas)/g;t.forEach((function(e){n=n.replace(e,"")})),n=n.replace(/\n+/g,"\n");var a=n.match(o),i=n.split(o).filter((function(e){return""!==e})).map((function(n,t){return[a[t],e.parseCategory(n)]}));i.forEach((function(n){n[1][0].forEach((function(t){var o=n[0],a=(o.includes("Cloning"),o.includes("Pseudomonas")?"blue":"red"),i=t[0].split(":")[0],r=t[0].split(":")[1].trim(),s=parseInt(i.split(".")[0]),c=parseInt(i.split(".")[1])-1;e.dates.push({dot:a,popover:{label:o},dates:new Date(2021,c,s),customData:{who:r,actions:t[1],method:o}})}))}))}},{key:"parseCategory",value:function(e){e=e.trim();var n=/(^|\n)\d+.\d+: [\w ,]+\n/g,t=e.match(n);t=null===t?[]:t.map((function(e){return e.trim()}));var o=e.split(n);return o=null===o?[]:o.map((function(e){return e.trim().replace("\n","
")})).filter((function(e){return""!==e})),[t.map((function(e,n){return[e,o[n]]}))]}}]),t}(g["d"]);Object(u["a"])([Object(g["e"])("date")],Fn.prototype,"events",null),Fn=Object(u["a"])([Object(g["a"])({components:{Calendar:yn["DatePicker"]}})],Fn);var Cn=Fn,Bn=Cn,kn=(t("205a"),Object(F["a"])(Bn,An,bn,!1,null,"052d9042",null)),Sn=kn.exports,Un=function(){var e=this,n=e.$createElement,t=e._self._c||n;return t("div",[t("div",{staticClass:"markdown",domProps:{innerHTML:e._s(e.htmltext)}})])},Dn=[],Mn=t("d4cd"),Pn=t.n(Mn),Nn=function(e){Object(h["a"])(t,e);var n=Object(d["a"])(t);function t(){return Object(c["a"])(this,t),n.apply(this,arguments)}return Object(l["a"])(t,[{key:"htmltext",get:function(){return(new Pn.a).render(this.mdtext)}}]),t}(g["d"]);Object(u["a"])([Object(g["c"])()],Nn.prototype,"mdtext",void 0),Nn=Object(u["a"])([Object(g["a"])({components:{}})],Nn);var Qn=Nn,Tn=Qn,Gn=(t("03708"),Object(F["a"])(Tn,Un,Dn,!1,null,null,null)),Ln=Gn.exports,Yn="# See below for the protocols used. *General* contains the larger scope routine methods, while the others are more specialised documents.\n## [General Protocols](#/ProtoGeneral)\n## [Protein purification under dentaturing conditions for medium level expression](#/ProtoHisTag)\n## [Medium-scale protein expression for purification](#/ProtoMediumProtExp)\n## [Protein re-folding parameters](#/ProtoProteinRefolding)\n## [Larger-scale protein refolding](#/ProtoProteinRefoldingLarger)\n## [Silver staining](#/ProtoSilverStaining)\n## [Small-scale protein expression](#/ProtoSmallProtExp)",xn='# Education & Communication \n\n \n\n## Overview: \n\nAs we began our design thinking process and tried to find out what the public knew about Per- and polyfluoralkyl substances (PFAS), we found that a lot of people knew next to nothing about PFAS or of the ramifications that comes with the use both for human health and for the environment. After we talked to experts within the field we felt that it was important not only to make a detector to be able to reduce usage of PFAS containing wax in skiing competitions, but also to inform the public about how common these faul substances are in our day-to-day life. \n\n \n\n \n\n## \\#PFASeOut \n\nJuly 2021 the UiOslo team challenged the public and other iGEM teams through instagram to go a week without PFAS containing products. We also encouraged through our promotion video with this challenge. \n\n \n\n\n\n \n\nThis was a challenge each one of us in the team took upon to do throughout the competition as well. The goal with this was to make people aware of how many different types of products actually contained PFAS and to educate them on what they actually put on and in their body. \n \n\nHere is how some of UiOslos team members challenge weeks went: \n\n \n\n### Helene’s PFAS-free week: \n\n \n\n“During the pandemic it was difficult to not use surgical masks, especially since this was enforced by the government when 2 meters distance could not be held. PFAS containing products were also used when a choice of wearing a raincoat, boots and other water repellent clothes vs getting wet and catching a cold was implemented for us. Fortunately we made sure our backing paper was PFAS-free and didn\'t microwave any popcorn. A problem occurred when most of us mostly only have teflon pots and pans." \n \n\n### Barathy’s PFAS-free week: \n\n“ I wanted to bake bread, and used baking paper that had the Nordic Swan on it to be sure it did not contain PFAS, but I did not think about the cake tin, which probably contained PFAS, because of its non-stick properties.” \n\n \n\n“Throughout this week I was curious about the grease-resistant properties of food packaging. I learned that a lot of fast food packages often contain PFAS. ” \n\n \n\n \n\n \n\n“Since I found out about this, I tried to make homemade fast food with kitchen utensils I was certain did not contain any PFAS, which was quite interesting and educational.” \n\n\n \n\n \n\n“When researching packaging I found out that a lot of disposable tableware, plates, cups has non-stick and grease resistance proparties from PFAS. I found out that there are Nordic Swan labels on PFAS-free products if there would have been a need to purchase disposable cutlery.” \n\n \n\n \n\n \n\n“I couldn’t find a picture, but since I was craving popcorn, I made popcorn in a pot without teflon or PFAS, since microwavable popcorn contains a PFAS lining in the bag. ” \n\n \n\n \n\n \n\n## PFAS Awareness Week \n\nSince we knew of other teams tackling PFAS-related issues, we reached out to all the teams we could find that worked with PFAS to collaborate to educate about PFAS. In the spring we had our first meeting with USAFA, and both teams realized we wanted to educate about the concerns around PFAS. We then got in contact with FCB-UANL as well. \n\n \n\nIn collaboration with the FCB-UANL and USAFA teams, our effort turned into PFAS awareness week, which took place 4th of October until 10th of October. This week we had an ongoing contest on Facebook and Instagram, where people were challenged to comment items they knew contained PFAS. The winner was announced at the end of the week and received a gift card from Akademika, one of our sponsors. \n\n \n \n\n \n \n\n \n\n \n\n \n\nThe main event of the week was an awareness panel on the 5th of October at 18:30 CEST facilitated on zoom. Our primary target audience were fellow students, regardless of their field of study, but the event was open for others as well. The reason for this was to engage peers and young adults in the PFAS discussion, as they are known to engage in social and environmental discourse from numerous reports about youth and volunteering from the Centre for Research on Civil Society and Voluntary Sector, which is a collaboration between Norwegian Research Centre and the Institute for Social Research (NORCE). One of the reports we based us on was called “Socially engaged youth - Participation in politics and organizational life among young people in Oslo” (2017). [1] \n\n \n\nAt the panel the participants got the opportunity to listen to an introductory talk from one of the UiOslo members, and then a longer talk on the topic from Line S. Haug, a senior scientist from the Environment and from the Norwegian Institute of Public Health, which is an agency under the Royal Norwegian Ministry of Health and Care Services, who was invited by the UiOslo team as a speaker. Then FCB-UANL presented about the iGEM competition and about their project and how it related to PFAS. Lastly UiOslo presented our project. At the end of the panel UiOslo announced the winner of the participation contest UiOslo had for the Norwegian panel participants, a competition where participants from Norway could win a gift card from Academica, a bookshop that sells academic books in Norway at the campus of the University of Oslo. The participants were from Ireland, Mexico, USA and Norway based on information provided by the participants themselves. \n\n \n\nThroughout the panel we got a lot of interesting inquiries to both the speakers and to the teams, regarding PFAS and our respective projects. Since it was on zoom the participants were encouraged to write in questions and discussion points in the chat feature, which made it easier to have a two way dialogue with the participants. \n\n \n\nWe recorded the panel to be able to watch back at it, and to share with the participants, the public and on our wiki page. The file was to large to include in the wiki, but we uploaded it on our [PFASeOUT Facebook](https://fb.watch/8NHuP9L3Vu/).\n\n\n \n\nAs a part of the marketing effort we made an informational and educational poster about PFAS, the awareness panel and an introduction to the iGEM competition. We printed up around 150 of them and distributed them around the University of Oslo’s different campuses and nearby metro stations. \n\n \n \n\n \n\n \n\n \n\n \n\n \n\n \n\n \n\n## Interview with Titan.uio.no \n\nAs a part of the outreach effort we were interviewed by Titan.uio.no about iGEM and this year\'s project. Titan.uio.no is a newspaper for science and technology at the University of Oslo, and is affiliated with the Faculty of Mathematics and Natural Sciences. The journalist wanted to get an overview of the project itself, what we did in the lab, our motivation and reason for our particular project and what made us sign up for iGEM in the first place. He also asked us if we could share our results and findings, so he could write about the conclusion of the project as well. \n\n \n\nPer the wiki freeze the article is yet to be published. The journalist that interviewed us estimated that it will be published closer to the Jamboree. \n\n \n\n \n\n## Blog article for Eurofins \n\nEurofins offered free of charge sequencing analysis in exchange for a blog article. Eurofins specializes in chemical, microbiological and sensory laboratory analyzes. Since Eurofins Scientific has branches in more than 50 countries, we will have a large outreach with this blog article. \n\n \n\nIn contrast to the Titan.uio.no article this was more in depth, with heavy emphasis on scientific communication going into more details of the compounds how it affects the environment and studies linking PFAS containing ski wax with health problems for the waxers. \n\n \n\nPer the wiki freeze the blog article is still under revision, and has yet to be published. \n\n \n\n \n\n## Speaking at lectures at the university \n\nBoth as a recruitment strategy for the next year\'s iGEM team and as an educational platform we attended multiple lectures at master level courses for chemistry and biology students. We presented about iGEM and the competition, our project and educated the students about the PFAS and its ramifications. \n\n \n\n \n\n## Social media \n\nWe have used our social media platforms to give informational posts with resources that the public could use to make well informed decisions around PFAS containing products in Norway. To mention some we shared educational videos, articles for the Royal Norwegian Ministry of Climate and Environment and from the Norwegian Institute of Public Health, which is an agency under the Royal Norwegian Ministry of Health and Care Services. \n\n \n\nWe also made a post about the Nordic Ecolabel or Nordic swan (norwegian: Svanemerket) which is the official sustainability ecolabel, introduced by the Nordic Council of Ministers, for all kinds of products for brands sold in the Nordic countries (Norway, Sweden, Finnland, Island and Denmark). The ecolabel is judged within criterias including health, environmental and quality arguments, and they, together with the EU Ecolabel, have strict guidelines for PFAS containing products such as: \n\nFood paper and baking paper \n\nDisposable packaging such as disposable cutlery, plastic foil, cardboard plates and cardboard packaging \n\nFurniture \n\nClothes and other textiles \n\nPaint \n\n \n\n\n \n\n \n\n \n\n## Outreach to the other norwegian universities \n\nWe knew early that we wanted to inform about the iGEM competition to other Norwegian universities, and to use this opportunity to educate students from the other universities about our project and about PFAS. Our plan was to have a visit and a meeting with the University of Bergen, University of Tromsø and The Norwegian University of Life Sciences. Only the University of Bergen showed their interest in such a meeting. \n\n \n\nIn collaboration with NTNU Trondheim we organized a meeting with the students and professors at the university. More information is available under [collaboration](https://2021.igem.org/Team:UiOslo_Norway/Collaborations). \n\n \n\n \n\n \n\n## PFAS labeling for consumers \n\nAs an effort to make it easier for the Norwegian population to make an informed decision when purchasing products we wanted to come into contact with the Royal Norwegian Ministry of Health and Care Services to make a dedicated PFAS-label to indicate whether a product contains PFAS or not. We contacted this ministry under the Norwegian government since they are in charge of health policy, health legislation, public health and health care services in the country. \n\n \n\nPer the wiki freeze we have yet to hear back from them. \n\n \n\n \n\n## What we wanted to accomplish, but was limited to \n\n \n\n### Movie night with a lecture and discussion panel \n\nEarly in our project research we discovered that there were some movies depicting PFAS and its dangers. “Dark water” was one such film. UiOslo thought it would be impactful to show this movie in the science library and have a lecture and discussion panel in addition to the viewing. We did try and research the information about how to obtain a Public Use Movie Licensing, but since there were so many unknown factors around actually being able to have a showing, we had to put the idea on hold. \n\n \n\n### Question booth around Oslo and on campus grounds \n\nThe UiOslo team wanted to set up a booth where it was encouraged to ask questions, inquiries and debate around the PFAS problem, and around regulations done in Norway and the EU. This came unfortunately not to fruition since it was difficult to plan due to the restrictions in Oslo. \n\n \n\n### Educational campaign with the Nordic Swan \n\nIn April we contacted the Nordic Swan to have an educational campaign to promote information around products containing PFAS. We hoped for a month-long campaign, but unfortunately we weren’t able to make substantial contact with the Nordic Swan to be able to host such a campaign with them. \n\n \n\n \n\n\n \n\n \n\n### References: \n\n \n\n[1] https://www.regjeringen.no/no/dokumenter/rapport-samfunnsengasjert-ungdom---deltakelse-i-politikk-og-organisasjonsliv-blant-unge-i-oslo/id2545619/ \n\n ',Wn="# Proposed Implementation \n\n \n\n## Introduction \n\nWith our project we wanted to develop a system that detects PFAS (Per- and polyfluoroalkyl substances), specifically in ski wax. This compound in ski wax has been banned by the International Skiing Association (Fédération Internationale de Ski - FIS) due to health risk and environmental concerns, but due to the lack of on-site testing devices this ban has not been effectively implemented.1 The consumers we are aiming for are therefore primarily from the skiing community. When working on the implementation of our detection system, we wanted to first and foremost focus on developing a robust and user-friendly system. The robustness is needed for the ban to be effective and fair for the athletes, and the device must be user-friendly so it easily can be handled by the organizers of the skiing competitions. It would be a bonus if the detection system also gave the results within a reasonable timeframe. \n\n \n\n \n\n \n\nOur primary goal is to generate a device capable of detecting PFAS traces in ski wax, but we are hopeful the design can be elaborated to a more general detection system for other products at risk of containing these toxic compounds. \n\n \n\n## Device Design \n\nOur testing system contain a dehalogenase gene that we have isolated and transformed into E. coli for expression. Dehalogenase is an enzyme that catalyzes the cleavage of a carbon-halogen bonds from a substrate.2 Since fluor is a halogen, this enzyme can therefore cleave of the fluorid ions in the fluorinated tail of PFAS. If the ski wax that are tested contains PFAS, the dehalogenase will cleave off fluoride from the structure and free fluorid ions will then be readily detected colorimetrically. \n\n \n\n \n\n \n\n \n\nIt would be favorable to have a device where the consumer only needs to dissolve the sampled ski wax with a solution that will be included with the device, followed by mixing it with a solution containing the protein. After a short time, a strip that could then be dipped into the solution and give a colorimetric change which indicates whether the PFAS is present or not. This kind of qualitative detection test would meet our goal of a user-friendly test that is easy to operate, with small chance of human errors. \n\n \n\n \n\n \n\nWe have not reached a point on our project where we are able to prove that that this testing device will work, as illustrated in figure 3. However, this is how an ideal testing device would be, based on our results and work. \n\n \n\n## Production of the device \n\nThere are several challenges associated with the production of this test, as of now these are some points that needs to be considered: \n\n* The production of the enzyme. In our project we expressed the enzymes in E. coli and later isolated them. The isolation has been challenging and would have to be significantly improved upon to make it efficient and cheaper. \n\n* Enzymes are sensitive, and a big hurdle is how they would be stored in the testing device and to stabilize them for longer periods of time. \n\n* Ski wax is hydrophobic and will require an organic solvent. Proteins are not too fond of organic solvents and can precipitate out under the wrong conditions. It is important here, through trial and error, to find the sweet spot for amount of organic solvent. \n\n* During skiing competitions, temperatures are most often sub-zero, hence we would have to consider how our protein would react and work under these conditions. \n\nOur wish is for the device to be cheap and easily accessible to anyone who wish to test a substance for PFAS. In that vision our production line would also have to be proportional to a market price. \n\n \n\n## User safety \n\nThe users would have to take the necessary precautions when using our test. \nThere would be safety precautions to take in relation to pH levels, solution containing enzymes and the final disposal when the solution contains cleaved PFAS compounds. \n\nIt is not highly dangerous solutions, so we think that a good way to tackle user safety is by including rubber gloves, safety instructions which are short and concise with explanatory pictures, and link to a video, which goes through the same safety instructions and user manual that comes with the test. \n\n \n\n## Disposal of the product \n\nWe do not wish to contribute to further contamination of the environment with PFAS-compounds. And as our test cleaves the C-F bond, the possibility is there for smaller PFAS compounds to be made, that can be more toxic than the start compounds. Therefore, a proper disposal service would have to be established. Where the tests were delivered back and disposed of in a fitting manner, so that no further PFAS were emitted. \n\n \n\n## Conceivable change \n\nWe believe that an on-site testing solution will enable more reactionary measures to be taken and a more efficient exclusion of malpractitioners. This will hopefully in turn have a domino effect on the athletes and casual skiers, which put a pressure on ski wax manufacturers to find new solutions to substitute PFAS in ski wax with a solution with the same properties that is more environmental- friendly and non-toxic. Innovation within this niche can also be applied to other consumer products as well, if a new chemical with the same properties is developed. \n\nAs a team we also have faith in consumers, specifically that a well-informed consumer wants to and will make the right decisions. Therefore, it is important to continue with the information campaign of PFAS to the general public. \n\n \n\n \n\n \n\n \n\nReferences: \n\nhttps://www.fis-ski.com/en/international-ski-federation/news-multimedia/news/flourinated-wax-ban-implementation-to-begin-in-the-2021-22-season \n\nKeiji Jitsumori, Rie Omi, Tatsuo Kurihara kurihara, Atsushi Kurata, Hisaaki Mihara, Ikuko Miyahara, Ken Hirotsu, and Nobuyoshi Esak (2020). X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from Burkholderia sp. Strain FA1. Journal of Bacteriology. \n\n \n\nAll figures on this page have been created with Biorender.com ",Kn="# Project Description \n\n \n\n## The problem: \n\nOver the last 50 years Per- and polyfluoralkyl substances, PFAS for short, has been used in a wide range of consumer products. \n\nThese compounds are a group of man-made chemicals, with over 7000 compounds, which are categorized according to their functional group, some examples being PFOA (R =COOH) and PFOS (R=SO3H). \n\n \n\n \n\n \n\n \n\n \n\n \n\nPFAS-compounds consist of a carbon chain of varying length which is saturated with fluoride, and at the other end there is a functional group which defines the properties of the compound. \n\nThe carbon-fluorine bond is considered to be one of the strongest bonds in organic chemistry, and as a result of this strength, PFAS compounds are highly stable. \n\nIn addition to their stability, they are amphiphilic compounds, which means they have a hydrophilic part (functional group) and a hydrophobic part (fluorine saturated carbon chain). \n\nIt is because of these properties that they are so well suited for many applications, some being: cosmetics, rain clothes, impregnation spray, fire retardants, surgical masks, cleaning products, food wrappings, pots and pans (non-stick), paint, ski wax, and many other products. \n\nPFAS compounds are unique chemicals with a wide range of useful applications that cannot be rivalled by any other known chemicals. \n\n \n\n## The drawback is that the properties that makes them so useful, is also what makes them so problematic. \n\n \n\n \n\n \n\n \n\nBecause of the stability of the C-F bond, PFAS are difficult to degrade, both chemically, biologically and physically. There is no known natural breakdown path in the environment, and because of their lipophilic nature these chemicals have a high potential for bioaccumulation and biomagnification. \n\n \n\nBecause of this, PFAS compounds have gotten the abbreviation “forever chemicals”. \n\nThey are spread through the environment, are present almost everywhere, and as a result of this, most humans have some concentration PFAS in their blood. \n\n \n\nSince there are over 7000 different PFAS compound, the research on possibly health effects is most comprehensive for PFOS and PFOA, the effects observed are as follows (https://www.fhi.no/ml/miljo/miljogifter/fakta/fakta-om-pfos-og-pfoa/): \n\nInhibits the production of antibodies after vaccination. \n\nInhibit fetus development \n\nToxic to the liver \n\nReduction in the metabolic hormones T3 and T4 \n\nPossibly carcinogenic \n\nIncreases the chance of acquiring diseases \n\n Several other possible health effects are also being researched at this time. \n\n \n\n## Our motivation: \n\nAmong all the things that contain PFAS – ski wax is one of them. \n\nTraces of PFAS have been found around skiing arenas from fluorinated ski wax [1]. In the long run this can have serious consequences on the eco-system surrounding the skiing tracks and can for example leak out into lakes and rivers surrounding the ski tracks. Since these chemicals cannot be degraded naturally, they bioaccumulate, and negatively impact marine- and freshwater ecosystem. They move through the food chain and ultimately end up on our dinner table. \n\nIn 2019 FIS (International skiing federation) banned the use of fluorinated ski wax from all their competitions, in relation to the scientifically proven detrimental health and environmental effects. \nThe ban has been postponed until after the 2021/2022 season due to the lack of a robust and user-friendly detection method [2]. \n\nEven though skiing competitions and the use of PFAS in ski wax is a small part of the global emissions of PFAS per year, it is a good starting point to tackle the global problem which is PFAS. \nA proper detection method would open for the penalization of users in a competition setting, possibly stop the production and sale of ski wax containing PFAS and reduce the release of PFAS to the environment, which is on average over 1 ton pr year [3]. \n\nWe found this problem highly interesting and saw the potential for this project to have far reaching effect into other areas where PFAS is being utilized. \n\n \n\n## Our solution: \n\nWe decided we wanted to create a robust, easy to use detection mechanism for PFAS in ski wax by utilizing enzymes called Dehalogenases that catalyzes the removal of a halogen atom from a halogenated substrate, producing free halogen ions such as fluoride [4]. This can be exploited by detecting electrochemical changes that occur in the presence of the fluoride ions. We envisioned a device where the enzyme could be immobilized, and ski wax could be applied, initiating a measurable enzymatic reaction that could produce a read-out. This would be a qualitative test, indicating if PFAS is present in the sample or not. \n\n \n\n \n\n \n\n \n\n \n\n \n\n References: \n\n \n\n[1]. Randi Grønnestad, Berta Pérez Vázquez, Augustine Arukwe, Veerle L. B. Jaspers, Bjørn Munro Jenssen, Mahin Karimi, Jan L. Lyche, and Åse Krøkje. Levels, Patterns, and Biomagnification Potential of Perfluoroalkyl Substances in a Terrestrial Food Chain in a Nordic Skiing Area. Environmental Science & Technology 2019 53 (22), 13390-13397 \n\nDOI: 10.1021/acs.est.9b02533 \n\n \n\n[2]. FIS. Update on FIS Fluorinated Ski Wax Ban [Internet]. [Updated 02.06.21]. Available from: https://www.fis-ski.com/en/international-ski-federation/news-multimedia/news/update-on-fis-fluorinated-ski-wax-ban \n\n \n\n[3]. PFAS in the treatment of skis — Use, Emissions and Alternatives. Miljødirektoratet/ Norwegian Environment Agency. Wood Environment & Infrastructure Solutions 2021. \n\n \n\n[4]. Keiji Jitsumori, Rie Omi, Tatsuo Kurihara kurihara, Atsushi Kurata, Hisaaki Mihara, Ikuko Miyahara, Ken Hirotsu, and Nobuyoshi Esak. X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from Burkholderia sp. Strain FA1. Journal of Bacteriology 2020. \n\n \n\n \n\nFigure 2 and 3 created with biorender.com \n\n \n\n ",zn='# Contribution \n\n## Support and sponsorship for future Norwegian iGEM teams \n\nWe had a meeting on the 23rd of August with the following companies where the iGEM competition was presented together with this year’s project. The participants were motivated to support future iGEM teams with knowledge and/or funding. \n\n* Inven2 - a Norwegian company that helps the scientist to develop their ideas and patent it) \n\n* The Life Science Cluster – A network for companies and organizations where life science is in focus. \n\n* Sharelab - Launchpad for start-ups, academics and companies that develop products and services using life science and biotechnology \n\n* The Centre for Digital Life Norway– National centre for biotechnology research, education and innovation \n\n* UiO:Life Science – Our university, UiO, has started up a part of the university with a focus on Life Science. They are currently building a new building only for Life Science. \n\n \n\nDigital Life Norway also resulted in sponsoring our iGEM project this year based on an application we submitted by the 20th of August. \n\n## Lab results that future iGEM teams can take knowledge from \n\n \n\nIn the lab, we learned that the Dehalogenase enzyme from Burkholderia cepacia and Pseudomonias aeruginosa when trying to express the proteins under our conditions the result was insoluble protein for the proteins from both bacteria. Which, as far as we know, was not known. This means the protein has to be purified under denaturation conditions followed by refolding. We found no data or literature about this in our research, so we had to test this out ourselves and future iGEM teams can take this into account if they want to express the proteins. The enzyme from Pseudomonias aeruginosa had the strongest band with the refolding condition 350 NaCl, while the enzyme from Burkholderia cepacia had the strongest band with 10% glycerol. To find different refolding conditions to try, we conferred with the iFOLDTM Protein Refolding System 1 from Novagen®. This system contains 92 different buffers with unique combinations of salts concentrations and pH. We picked out a few alternatives that were available in the lab. Tris-buffer was chosen because it does not contain salt, as opposed to PBS buffer which already has a NaCl concentration of 137 mmol/L. \n\n### NaCl variations in concentration with Tris-buffer. \n\n* 0 mM NaCl \n\n* 150 mM NaCl (this is similar to what would be the natural concentration in a cell) \n\n* 350 mM NaCl \n\n \n\n### Other variations with Tris-buffer \n\n* Glycerol (10%) \n\n* 500 mM Arginine \n\nThese are conditions are based on refolding conditions that have been shown to work experimentally. If your team experiences little to no result after trying these refolding conditions, we would recommend trying the pH variations for 7, 7.5, 8 and 8.5. \n\n \n\n## Parts \n\nWe added two parts to iGEM, `BBa_K3709000` and `BBa_K3709001`. \n\nBBa_K3709000 is the DNA sequence coding for the dehalogenase enzyme from **Burkholderia cepacia** \n\nBBa_K3709002 is the DNA sequence coding for the dehalogenase enzyme from **Pseudomonias aeruginosa** \n\n## Inspiring for more participants and teams in Norway \n\nLast year’s team for UiOslo only had 3 participants, and this year only had 6. Even though we have worked well, we did notice our limitations because of our number. Therefore, we have tried to inspire students at our university to join both this and next year’s team. \n\nFrom May till October, we’ve posted about iGEM in several student groups for the students at the University of Oslo. We also had two meetings with pharmacy students to try to recruit students from outside of the Department of Bioscience at the University of Oslo. \n\nTogether with NTNU Trondheim, we had a meeting with students from the University of Bergen, you can read more about this under the collaboration. We did this to inspire more universities in Norway to have teams in iGEM. There will also be a meeting with a possible supervisor for this University after the wiki freeze. \n\n \n\n## Making wiki easier for future teams \n\nWe developed a framework for pushing an iGEM MediaWiki- site using modern web tools (specifically vue). This will enable future smaller teams with marginal technical expertise to focus on the biology part of the competition and push their contents, results and statuses with ease, while enabling more advanced users to engineer cutting edge ui-tools, and optionally deliver advanced software interactively through the iGEM web page. \n\n ',En='# Engineering success \n\n \n\n \n\n \n\n|Figure 1: Illustration of several design-build-test-learn-improve-cycles we have been through during this project. \n\n\n## Introduction to the problem \n\nWe wanted to develop a testing device to detect PFAS in ski wax. In 2021, International Ski Federation (FIS) announced a ban on the use of ski wax containing per- and polyfluoroalkyl substances (PFAS) during competitions. (FIS 2021) For now, no reliable detection device exists that can test the skis of athletes on-site. We talked to the Norwegian Ski Association about how they would want such a test device to be designed for it to be implemented in competitions. They needed a way to test the wax under the sole of the ski without damaging the applied wax, they wanted a device that could give results in a short amount of time. The goal was to test every ski both before and after the competition and have a result ready before the winner enters the podium, and for that we needed a system that was able to degrade PFAS chemicals into something that can readily be detected, like fluorides. One of PFAS characteristics is their carbon-fluorine bonds. If we manage to break this bond, free fluoride ions will be present. These ions can easier be detected by available testing mechanisms that exploit the electrochemical changes in the presence of fluoride ions. To break this carbon-fluoride bond using synthetic biology, the appropriate enzyme is needed. In our project we have therefore been searching for the correct enzyme that can carry out the desired process and clone the gene into E. coli to express and purify the enzyme, which is a dehalogenase, an enzyme that catalyzes the removal of a halogen atom from a halogenated substrate. (Jitsumori et al, 2020) We identified the necessary enzyme, a dehalogenase, that could catalyze the removal of a halogen atom from its halogenated substrate. For this purpose, we decided to clone the necessary gene and express and purify the protein in E. coli cells. \n\n## Design of the project \n\nWe started our project by looking at previous iGEM teams to investigate if someone had done something similar. The USAFA iGEM team from 2019 had made a reporter system using a gene called prmA from the Rhodococcus strain RHA1, which expression level increased in the presence of PFAS. [Team USAFA] (https://2019.igem.org/Team:USAFA/Description) Unfortunately, due to shipping restrictions, we could not get hold of the strain. Therefore, we proceeded with another alternative approach, namely detecting the degradation of PFAS in the form of fluoride ions, using a Dehalogenase enzyme from both Burkholderia sp. FA1 and Pseudomonas aeruginosa strain FA1 (Sunanthaa et al., 2020). \n\nBoth P.aeruginosa PAO1 and Burkholderia sp. are biosafety level 2 (BSL2) pathogens and are therefore not practical to work with in the lab, while E. coli bacteria that is much more convenient and safer to use. Therefore, the sequence for the dehalogenase from Burkholderia ordered codon optimized for expression in E. coli as a gBlock from IDTDNA with a hexa His-tag and a cleavable tag. \n\nThe dehalogenase from P. aeruginosa PAO1 we could isolate from genomic DNA we had available at the lab. This gene would be amplified using PCR. \n\nAfter we had successfully cloned the gene of interest into the pBAD expression vector, our plan was to transform competent Escherichia coli BL21 cells with our construct and induce expression of the enzyme in E. coli. \n\nThe other part of the design of the project was to decide on what the actual testing device would be. We had a meeting with representatives from companies working with innovation in research, and they expressed excitement about the idea of a testing device based on the lab-on-a-chip-technology. With this kind of a device, we envisioned that the purified dehalogenase enzyme could be applied together with samples from ski wax. From there, electrochemical signals could be measured from the free fluoride ions that would be formed from the cleavage of fluor-carbon bonds. \n\n \n\n## Build, test, learn and improve \n\nOur first step into realizing our detection mechanism was to amplify the correct gene from both bacteria strains Pseudomonas aeruginosa PAO1 and Burkholderia sp. To amplify the desired genes, we designed the suitable primers for both strains and amplified by using PCR. The PCR products were then loaded onto a gel electrophoresis to validate if the right genes have been amplified, thus the size of the desired genes are known. The gene from Pseudomonas aeruginosa was troublesome to amplify. We had several steps of improvements before the cloning was successful (see illustration). \n\nThen we could proceed with expression, which also proved to be a challenge, but also this challenge we overcame going through trial and error in the lab. We started by lysing the cells mechanically using zirconia beads and a bead beater but switching to chemical lysis using a detergent streamlined the lysis process to a large extent. The expression protocol was successful, the protein was expressed. However, another problem arose; the protein was expressed in the insoluble fraction, most likely in inclusion bodies. \n\n \n\n### PURIFICATION \n\nTo purify the protein, we used immobilized metal affinity chromatography (IMAC) with nickel NTA (Ni-NTA) resin. For this purpose, a poly-histidine tag was attached to our desired gene during the cloning process. Based on Coomassie and silver staining after electrophoretic separation of the expressed proteins on a SDS page, we discovered that the purification was not optimal. Several other undesired bands on the gel indicated that the product was not pure enough. We optimized this step by increasing the frequency of the washing step and the volume of the washing buffer. We ensured that all the unwanted proteins that did not bind to the Ni-NTA matrix were washed away and removed by measuring the Absorbance at 280nm of the washing suspension after each wash. When the Absorbance at 280 nm of the washing fraction was at the background level, we stopped the washing steps to prevent washing away the desired proteins that were bound to the Ni-NTA matrix. A new Coomassie and silver staining indicated that the impurities were still present, but at a lesser extent. \n\nFor further optimization, we decided to scale up protein expression and for that purpose use an FPLC system (BioRad) for the needs of the protein purification step. \n\n### REFOLDING \n\nOur protein was found in the insoluble fraction, which might be due to the generation of inclusion bodies, as mentioned earlier. For this reason, our protein was purified under denaturing conditions. In order to obtain folded and functional protein, we prepared refolding buffers. Five different refolding buffers were prepared and tested out. \n\nTo find different refolding conditions to try, we used as a guideline the iFOLDTM Protein Refolding System 1 from Novagen®. This system contains 92 different buffers with unique combinations of salts concentrations and pH. We picked out a few alternatives that were available for us in the lab. We used a Tris-based buffer and we decided to use different salt concentrations as shown below. \n\n \n\n### NaCl variations in concentration with Tris-buffer. \n\n* 0 mM NaCl \n\n* 150 mM NaCl (this is similar to what would be the natural concentration in a cell) \n\n* 350 mM NaCl \n\n \n\n### Other variations \n\n* Glycerol (10%) \n\n* 500 mM Arginine \n\nThese conditions are based on refolding conditions that have been shown to work experimentally. \n\nThe refolding conditions that appeared to be most efficient for the dehalogenase from Burkholderia was Tris + 10 % Glycerol. The protein from P. aeruginosa indicated refolding in Tris + 350 mM NaCl. \n \n## TESTING DEVICE \n\nAs for the lab-on-a-chip idea, time proved to be against us. We had to revise our original plan as it was too ambitious to develop in the scope of the iGEM competition. As a first step towards a detection mechanism, we acquired detection strips that will change color if fluoride ions are present. We decided that this would be a good starting point from where we could further develop the testing device into a lab-on-a-chip. \n\nWith everything set in place we identified a couple of colorimetric detection systems that would allow us to monitor fluoride ions released upon catalytic cleavage of a fluorinated substrate by our enzymes of interest. As a proof of principle and as a positive control we measured the presence of fluoride ions in a NaF solution at various concentrations. Based on this knowledge we will dissolve our PFOA compound into a relevant concentration that should be within detection limits of the colorimetric detection kit if our enzymes are able to perform the intended reaction. \n\n \n\n## References: \n\nFIS (2021) Update on FIS Fluorinated Ski Wax Ban: https://www.fis-ski.com/en/international-ski-federation/news-multimedia/news/update-on-fis-fluorinated-ski-wax-ban   \n\n \n\nKeiji Jitsumori, Rie Omi, Tatsuo Kurihara kurihara, Atsushi Kurata, Hisaaki Mihara, Ikuko Miyahara, Ken Hirotsu, and Nobuyoshi Esak (2020). X-Ray Crystallographic and Mutational Studies of Fluoroacetate Dehalogenase from Burkholderia sp. Strain FA1. Journal of Bacteriology. \n\nGanesan Sunanthaa, Namasivayam Vasudevan (2020). A method for detecting perfluorooctanoic acid and perfluorooctanesulfonate in water samples using genetically engineeredbacterial biosensor. Science of the Total Environment. \n\n \n\n ',On='# Collaborations we created \n\n## PFAS awareness Panel \nWe set up a PFAS awareness Panel with the iGEM teams [FCB-UANL](https://2021.igem.org/Team:FCB-UANL) and [USAFA](https://2021.igem.org/Team:USAFA) to create a digital meeting open to everyone. Line S. Haug was invited as a speaker by us, who works as a senior scientist at FHI (The Norwegian Institute of Public Health is a government agency under the Ministry of Health and Care Services in Norway). She has long experience with PFAS and had a lecture about and how it is in cross country skiing. There were 28 participants from Ireland, Mexico, Norway and USA. Our team, UiOslo, presented what PFAS is and our project. FCB-UANL presented what iGEM is and their [project](#sources). \n \n\n## iGEM information \nThe only iGEM teams in Norway is [NTNU Trondheim](https://2021.igem.org/Team:NTNU-Trondheim) and UiOslo, therefore we set up a meeting to recruit the University of Bergen by arranging a small talk over zoom with RU (Realistutvalget). RU is the student board where all students at the Faculty of Mathematics and Natural Science at University of Bergen are represented. Here an introduction of what iGEM is and what NTNU and our team’s project was this year with the goal to spread the word and have more participating teams in Norway. \n \n\n# Collaborations we’ve participated in \n\n## Kampen mot bakteriene – The war on Germs \nThe iGEM team [CCU_Taiwan](https://2021.igem.org/Team:CCU_Taiwan) made a cartoon where we supplied the translation of the book into Norwegian (bokmål). \n\n\n## iGEM Monument Challenge \nThe iGEM Monument Challenge was set up by the iGEM team [GO Paris-Saclay](https://2021.igem.org/Team:GO_Paris-Saclay) and posted on our instagram and facebook-page.\n \n\n## iJET \n\n[Team Darmstadt](https://2021.igem.org/Team:TU_Darmstadt/) Continued the iJET challenge from 2020, where the idea is to unite as many iGEM teams as possible in one video in a fun and creative way. ',In="",Rn='# Human Practices \n\nPer- and polyfluoroalkyl substances (PFAS) bioaccumulates due to the stability of its chloride-fluoride bonds, which are difficult to break down. Long term exposure to PFAS has been linked to high cholesterol, pregnancy-induced hypertension, thyroid disease, testicular cancer, kidney cancer and decreased response to vaccines [1]. PFAS is used in several consumer products due to its stain and water repellent properties. \n\n \n\n![X Figure 1: Illustration of a PFAS compound with C8-chain and potential functional group R.] \n\n## How it all started \n\nWhile deciding on a project, we found several articles regarding the negative health effects from long exposure to PFAS compounds. As none of us were aware of this, we found it eye opening. This revelation motivated us to focus our project upon PFAS, with the wish to try and contribute to minimize the use and the exposure. \n\nOut of curiosity we contacted a few people in our own social group and families to see if they were as unaware about PFAS and its effects as us. The result was jarring – no one knew what PFAS was or how it was used. It made us realise that the lack of information among the general public was a big part of the problem. So as a result, a large part of our project was centred around educating the public so that they could use their power as a consumer to put pressure on companies using PFAS in their products. This was done through our social media, with PFAS awareness week and our PFAS panel (for more details see our [collaboration](https://2021.igem.org/Team:UiOslo_Norway#/collaborations) and [partnership page](https://2021.igem.org/Team:UiOslo_Norway#/Partnership). \n\n \n\n \n\n## Reaching out to experts \n\nTo gain more knowledge about PFAS we had a meeting with **Katrine Borgå**, an ecotoxicologist at the University of Oslo. Her research is about understanding the processes and mechanisms for contaminant distribution, accumulation and what effects this has on the environment. During this meeting Katrine presented several research results on PFAS, including negative impacts on our health and accumulation in the environment. She made a good point that without the consumers support the producers would not make products containing PFAS. \n\nHer presentation and our discussion at the end of the meeting showed us a bigger picture of the problem. The research results she presented underpin the urgent need of better regulation of these chemicals. We learned a lot about the negative impacts of the human health and the environment that we were not aware of. This led to a discussion on the lack of responsibility placed on the manufacturers, and how many of these manufacturers mislead their consumers by the inaccurate labeling of products containing PFAS. We realized after this meeting that this problem is not widely spoken about enough in the public and decided that we wanted to change that by making a big part of our project about spreading awareness regarding this chemical. This meeting was quite valuable for the starting phase of our project. \n\n \n\n## Integrating the information \n\n \n\nAfter the meeting with **Katrine Borgå**, we gained new perspectives regarding the extent of the negative impacts of PFAS. We felt it was unethical to not convey this important message to make more people aware of this problem, therefore one of our main goals on this project was therefore to **spread awareness** about this problem. This was done through our PFAS awareness week, in collaboration with [USAFA](https://2021.igem.org/Team:USAFA) and [FCB-UANL](https://2021.igem.org/Team:FCB-UANL). In addition to this we have also sent an email to the Ministry of Health and Care Services in Norway where we emphasized the importance of clear and concise markings of products containing PFAS, so that the consumers can make an informed choice based on facts. \n\nThe only marking of PFAS-free products in Norway are the Nordic Swan Ecolabel. The Nordic Swan Ecolabel was established back in 1989 and is still used by the Nordic countries. This marking is used as a tool for the consumers, so it is easier to choose environmental-friendly and non-toxic products. We tried to contact the Nordic Swan Ecolabel back in April, as we thought their knowledge would be a great input for our project, but without luck. \n\n \n\n|Figure 3: Illustration of the nordic ecolabel [2]\n\n \n\n \n\n \n\n \n\n(Heading says: Deeply worried about fluor blind test – testing device analysed wrong) \n\n \n\nWhen deciding on a project we wanted to do something local, which could engage the public. We read about a ban for using PFAS containing ski wax, but that the implementation was delayed in the 2020/2021 season due to the lack of proper testing devices that can be used specifically during skiing competitions [3]. \n \n\nSkiing is a big part of the Norwegian culture, and most Norwegians have a relationship with winter sports. Since it is such a big part of our identity, we believed that this could be a problem that had the potential to engage people from all age groups. \n\n \n\n## Including potential users in our product development \n\nSince we looked into the idea of developing a detection mechanism for PFAS in ski wax, we wanted to hear some thoughts regarding this idea and the feasibility of the project from the skiing community. We therefore invited **Skiforbundet** (Norwegian skiing association) for a meeting. \n\nTwo specialists within ski wax appliers and a technical sport chemist from Skiforbundet joined us for the meeting. During this meeting we got a better understanding of the ski wax situation in the skiing community and why no actions have been taken since the ban was officially announced in 2019 by FIS (The international skiing association – Fedération Internationale de Ski). We learned more about the existing detection mechanisms that they have tried out and looked into, and why they do not satisfy their needs regarding testing of PFAS in ski wax. From this meeting we therefore got a grasp of what to focus on when designing a detection mechanism specifically for use during skiing competitions. \n\nThey informed us that the skier would be faster with the use of PFAS containing ski wax compared to a skier using performance-enhancing drugs. Which is why, without a proper detection method, most teams cannot afford to not use PFAS containing ski wax, as they would surely do significantly worse than their competitors that is using it. \n\nOne of the members from Skiforbundet stated that there will be no innovation without the development of a functional detection device for ski wax during the meeting. This made us realize the important impact of such a detection device, which will put the needed pressure on ski wax manufactures to find ways to develop new and more environmentally friendly products in the future. \n\nThis meeting with Skiforbundet was a huge success. We were asked important questions that we had yet thought about and they touched upon subjects that were relevant for us, which drove our project in the right direction. At the end of this meeting, we also got the contact information for relevant persons from SWIX (one of the largest skiing equipment manufacturers in Norway). \n\nSwix informed us that ski wax is only responsible for a small share of the total PFAS emitted every year. This was confirmed in a report by Miljødirektoratet, a governmental agency under the Ministry of Climate and Environment in Norway [4]. Their focus is primarily to reduce greenhouse gas emission, manage Norwegian nature and prevent pollution. Despite the amount, it is still PFAS being released, and there are good waxes on the marked that do not contain PFAS. To use PFAS in ski wax is not essential, and can these waxes can be removed from the market without any repercussion upon society. \n\nIf we were to create a usable detection method during this project, it would assist in phasing out the use of PFAS-containing wax entirely from this sport, which inspired us to name our project “PFASeOut”. \n\n \n\n \n\n \n\nDuring our meeting with Norges Skiforbund (The Norwegian Skiing Association), and through several mail exchanges with them, we got input on what type of test was desirable. It was not important for them for the detection device to measure PFAS concentration as long it could robustly detect PFAS in the ski wax. The test would ideally not be too expensive, so that skiing competitions for youth and children could also afford to buy the test. They also had a desire to have a time efficient test, so that it could be used during competitions. \n\n \n\n## Integrating the feedback \n\nAt this point we also considered making a biofilm that could be placed at the starting point for the competition, leaving an indication if a wax containing PFAS had been used. But this could potentially damage the wax and skiis of other skiiers. And it could possibly result in an emission of biohazardous waste into nature, as we are trying to reduce the emissions, this would be counterproductive. \n\nWe were also invited to test out our detection mechanism at skiing competitions in the 2021/2022 season to test out the device. \n\n \n\n \n\n \n\n## Feedback from potential investiors and entrepreneurs with implementation \n\nOn the 23rd of August our team had a meeting with companies that were entrepreneurs and/or potential sponsors who could give us feedback from their perspective. These companies were: \n\n* Inven2 - a Norwegian company that helps the scientist to develop their ideas and patent it \n\n* The Life Science Cluster – A network for companies and organizations where life science is in focus. \n\n* Sharelab - Launchpad for start-ups, academics and companies that develop products and services using life science and biotechnology \n\n* The Centre for Digital Life Norway– National centre for biotechnology research, education and innovation \n\n* UiO:Life Science – Our university, UiO, has started up a part of the university with a focus on Life Science. They are currently building a new building only for Life Science. \n\n \n\nThe feedback was positive to biosensors, which is why we also considered this for a long period of time, and they also gave feedback to focus on the cost of each test. It had to be cheap to produce and easy to use! This was implemented further when developing the product. \n\n \n\n## Conclusion \n\nBy working with potential users, investors and experts in the process we were able to design a user-friendly product. Our education and communication focus on PFAS was directly linked to what we learned from our meeting with experts in this field that we met with along the way. \n\n \n\nReferances: \n\n1 https://www.fhi.no/ml/miljo/miljogifter/fakta/fakta-om-pfos-og-pfoa/) \n\n2 https://www.nordic-ecolabel.org/ \n\n3 https://www.nrk.no/sport/dypt-bekymret-etter-fluorblindtest-_-testapparat-analyserte-feil-1.15419384?fbclid=IwAR1VCq6riunVxqjblLKW_KBMZrOinLYabwAodJdzMfM1VMtIVCpMduVBURs \n\n4 https://www.miljodirektoratet.no/aktuelt/fagmeldinger/2021/mai-2021/ny-rapport-om-miljogifter-i-skismoring/ \n\n ',jn='# Partnerships\n## NTNU \nSince NTNU Trondheim and UiOslo are the only two teams participating in iGEM in Norway we had contact from early on. We worked as a support group for each other, sharing good and bad experiences so that both teams could gain knowledge and save time because of the other team’s experience. \nWe hoped to be able to have a meeting in person, but the pandemic made it difficult. What we did have was several meetings online between the two teams, where some included the supervisors as well. \n\n## Learning through the other team\'s experiences \nNTNU Trondheim shared their experiences and research about biosensors, which was something that triggered an interest in our team. A biosensor was highly considered in our detection part due to their presentation. We ended up going with something else, but we highly recommend trying out a biosensor if a team would be inspired to build upon our project in the future. \nWe both shared our plan for promotion movies in a meeting, where NTNU Trondheim went with animation while we went with filming at SNÖ and skiing, sharing that we had asked for sponsorship for access to the facilities to film for the movie. \n\nNTNU also shared their sponsorship approach with us, which inspired us. They had contacted both related and unrelated potential sponsors with great success. They used gold, silver and bronze levels. We tried the same but did not end up with the same result. Our teams found out it worked better for us to seek out specific sponsors and have a meeting with them to tell them about us, our project and iGEM. The shared experiences were useful for both this year’s teams and future ones in Norway, as both of the teams ended up needing to go with different approaches for a good result. We also told them about an application for iGEM teams in Norway so that they also had an opportunity to apply. \n\n\n\nWork with Wiki\nDuring the summer our two teams supported each other with the Wiki when problems arose. Tips and experiences about the MediaWiki were exchanged, especially around how the setup works and how web frameworks could be used. The web team of NTNU and our own discussed in detail technical solutions, and critical shortcomings were elucidated, among others regarding deliverable URLs. Equivalently, code optimisation tips were shared in both directions, and discussions on how to override and work with the existing MediaWiki stylesheets benefited both teams.\nDue to our partnership, both teams had more efficient coding with the wiki and website display, and both teams were available for support for each other on the wiki during the whole competition. \n\n\n\n\n## Recruiting more iGEM teams in Norway \nOn the 11th of October, we invited NTNU Trondheim to a meeting our team had set up with Realistutvalget (Representation of all the studies at the faculty of Mathematics and Natural Sciences) at the University of Bergen. There we presented iGEM, this year’s project from NTNU and UiO. The goal was to recruit more teams in Norway for the competition with the approach that all students, no matter the background, would be greatly valued in the competition. We hoped the presentation would inspire the students to request the possibility to compete, where the two teams would support them in the start-up years. Both teams answered RU’s questions and gave them contact information for future contact. \nOur team also contacted a few professors at the University of Bergen. The plan is to set up a second meeting with the professors to present the same presentation from both teams and answer questions, but this might occur after the wiki freeze and iGEM 2021. \n\n## Through thick and thin \nWe invited The NTNU team to our PFAS awareness panel, where they had one representative and also shared information about the panel through their social media accounts. \nCommunicating regularly with another team, hearing about their ups and downs, were comforting and motivating. If there was a problem, we knew there was always a possibility to check with the other team if they were struggling with the same issues or had a solution, this was good to have when a deadline or problem approached. \nAfter the summer our focus was a feedback-based partnership, where we got input on our illustrations for the PFAS conference and we gave input on their abstract and title. \nA partnership does not end even though we have had wiki freeze. At the beginning of November we’re planning on running through our presentations for each other and give feedback so that both teams will be better prepared on the day we will present. \nHaving this type of partnership is highly recommended. It was easy to ask each other for tips, tricks, advice and input. Sometimes it’s comforting just to not feel alone, which has been a large part of this year with the pandemic and its limitation.\n\n\n## PFAS collaboration \nWe realised we had a very similar project with USAFA and FCB-UANL when we first had decided to work with PFAS. We tried early in the process to reach out to USAFA and FCB-UANL as they had worked with PFAS-related projects. \n \n## Spreading the word about PFAS \nAfter our first meeting with USAFA in springtime, we realised that all teams had an interest in educating the public about PFAS. A children cartoon was among ideas that were discussed, together with our social media challenge to live one week without PFAS containing products. Our communication continued from this point, and we joined forces together with FCB-UANL to plan a PFAS awareness week ending with a panel. \n\n\n\n\nOn our side, we printed out 150 posters which we hung up around campus about PFAS, iGEM and the panel. Our team also set up the Facebook event, sent a mail to HQ iGEM about the event and created the graphics that were shared on the Facebook event. UiOslo also invited Line S. Haug, a senior scientist at FHI (The Norwegian Institute of Public Health is a government agency under the Ministry of Health and Care Services in Norway). She has long experience with PFAS and had a lecture about and how it is in cross country skiing. \n\n\n\nOur team contributed by having a short introduction of what PFAS is and the possible negative health effects, while FCB-UANL presented what iGEM is. Both UiOslo and FCB-UANL shared their project for this year and answered questions asked in the chat. All three teams (FCB-UANL, UiOslo and USAFA) made their graphics they could share, and we tagged each other in posts during the PFAS awareness week to involve and engage more people. All three teams assisted in planning the week and successfully carry out the ideas we had from the first meeting together. \n\n\n\n\n## Information-flow \nWith USAFA we shared what bacterium we used this year, and they told us they were planning on using 7 different ones. \nEarly on it became clear that we had researched the same literature. When choosing methods, we communicated with USAFA when we found out that they planned on using PerfectION Fluoride Electrode and Mettler Toledo SG78-FK2 F-ion probe. Due to this communication, these two options were considered, but we ended up going for a colourimetric assay as our focus was to make the test accessible in the long run. But these are options that could be considered by future teams that are interested in doing something related to our project.\n\n',Jn='\n# Human centered work\n\n## Meetings with experts – Input on design \n\n### Goal: \n\nTo design a device that meet the consumer’s needs and to address the right problems, we reached out to experts within the relevant field, including the national skiing association, ski wax manufactures and researchers. The purpose of these meetings was to present our idea and get input on the project by the expert’s knowledge and expertise. We also wanted to use these meetings as a platform to discuss and trouble shoot during our project, if we encountered on unexpected problems along the way.\n\n### Results: \n\nDuring our project we met with several experts within different field that were relevant for the development of our device. We found these meetings quite helpful, and with the input and knowledge we got from the experts, we got to see the problem we wanted to tackle from different perspectives. The meetings also served as trouble shooting and discussion sessions, along our process. \nIn the early phase of the project, we had a meeting with Katrine Borgå, an ecologist at UiO, which expertise is within contamination distribution and accumulation in the environment, including PFAS. From this meeting we were introduced to the negative impacts of PFAS, both on our health and environment. Katrine also elaborated on the use and labeling of these chemicals in the industry and how they prevent the national regulation we have on these kinds of chemicals in Norway.\nTo get to know the problem better and what to consider while designing our detection system, we were eager to talk to experts from the skiing community. We therefore arranged a meeting with the Norwegian skiing association (Skiforbundet) with two of their specialists within ski wax appliers and a technical sport chemist. During this meeting we got a better understanding of the ski wax situation in the skiing community and why no actions have been taken since the ban was officially announced in 2019 by FIS (The international skiing association – Fedération Internationale de Ski). We learned more about the existing detection mechanisms that they have tried out and looked into, which have not satisfied their needs. From this meeting we therefore got a grasp of what to focus on when designing a detection mechanism specifically for use during skiing competitions.\nSince SWIX is one of the largest manufacturers of skiing equipment, including ski wax, in Norway, we got in contact with them to get the specific details on the content of ski wax. \nThe experts that we met with along the way asked us important questions that we had yet thought about and questioned our project in ways that helped us shape and drive our project in the right direction. Most importantly they provided us with relevant information that helped us flag potential pitfalls and improve our design. \n\n### Future directions:\n\nIt is not always easy to do everything correct the first time around. We have therefore noted down a few points that we learnt after attending these meetings with experts, that might be valuable for us later or future iGEM teams. \nSomething we realized quite late, that would have been quite helpful for our project, is how to arrange a meeting with experts. Often, the company or department that you request a meeting with, would not mind if you have a request on who you want to meet up with. We have noticed that it is a huge advantage to meet with an interdisciplinary team. Of the larger companies and departments that we have met with, they have employees that with different expertise in a specific area. When having a meeting with such an interdisciplinary team, each expert will bring their piece of knowledge and advice, and you will therefore be able to investigate the problem from different possible angles and get a more complete grasp of it. \nThroughout the project we also experienced the difficulty of getting in touch with big companies that we wanted to arrange a meeting with. However, the experts that we did get in contact with often had connections with people from large companies within this field, which we got the contact information directly from. This is valuable information that which we would have taken advantage of more often if we knew about it earlier.\n\n## Education and public engagement\n\n### Goal: \n\nA central part of our work, beside from developing the PFAS detection system, is the passing on of knowledge. PFAS has many negatively documented health and environmental impacts. Still, the awareness regarding this subject is limited. PFAS are not only found in ski-wax, but a large variety of everyday items, ranging from pans and wrapping paper to microwave popcorn bags. As a team we have faith in consumers, specifically that a well-informed consumer wants to and will make the right decisions. To empower consumers of ski wax and other PFAS-containing products, we therefore want to promote our project to raise awareness and inform of the dangers of PFAS.\n\n### Results:\n\nTo reach out to the public, we published information regarding PFAS on several different platforms, including Facebook, Instagram, the university\'s home page and our Wiki page. The posts were mainly short and informative, but also included updates on the project’s process. On both Instagram and Facebook, we have over 700 followers, which we reach through with our posts.\nTo engage the public, we also held a competition on social media, where a lucky participant could win a prize. To attend the participant had to mention one thing that contain PFAS. The thought of this competition was to engage our followers to do brief research on PFAS to get an understanding of what it is and what usually contains PFAS, to be able to attend the competition. We also used this competition to promote our PFAS conference that we held the same week to promote our project and inform about PFAS. For this conference we invited Line Haug, an expert within exposure characterization and human biomonitoring of organic pollutants, as a guest speaker. She educated us on the hazardous impact of PFAS and presented results from her department’s research within this field. The participants also had a great discussion with her at the end of the presentation. There was a total of 28 participants on our PFAS conference. \n\n### Future directions:\n\nAs a future direction we hope to more effectively reach out to a larger audience, so even more people will learn about PFAS. To do so, it might also be beneficial to find a better method to engage the public.\n\n## Project presentation for entrepreneurs \n\n### Goal: \n\nOur team held a meeting with experts in entrepreneurship and innovation to get input on how our project could be developed further as a commercial product. This would also give us an indication of how our idea would be met by the public, in the eyes of people that have many years of experience helping start-ups. \n\n### Results:\n\nFor this meeting we invited several innovative companies and departments, including INVEN2, The Life Science Center, Share Lab, Life Science Center and relevant persons from academia. After presenting our project we got constructive feedback on the technical part and the feasibility of our project. The participants came with many great inputs on how we could improve our detecting system to build a better business case that would target the consumers expectations and needs. We also discussed the idea and process of patenting the detection system. \nConsidering the participants’ experience on hearing idea pitches from start-ups, they also gave us many useful tips on how to improve our presentation and presenting skills. \n\n### Future directions:\n\nFor future pitches and presentations of our project, we have learnt to structure our presentation in a more ordered manner and elaborate more on a few things that the participants felt we were lacking in our presentation. When presenting this PFAS problem, we should avoid exaggerating the problem and clearly state our findings, to avoid misunderstandings.\nSince we are aiming for this detection device to be implemented as a commercial product, we should also look more into the details regarding the implementation of this product in the future, particularly the actual production of the protein for our detection device and the production cost. \nMedia coverage – Interview with Titan and blog article for Eurofins\n\n### Goal: \n\nTo make people aware of PFAS in products we rely on every day, and encourage to choose PFAS-free products, we wanted to reach out to a broader audience to inform the public about PFAS and our project. Achieving media coverage will help us spread our message to an audience we would in other ways not be able to reach. Getting the message conveyed through a well-known media house or company will also help us establish a form of credibility.\n\n### Results:\n\nEurofins Scientific offered us sequencing analysis services in exchange of a blog article for their "DNA Universe blog". This was an excellent offer for us, thus Eurofins Scientific is an internationally recognized company that provide laboratory services and equipment, which reach out to an enormous group of people within the field of biology. We wrote a blog article that explained the negative impacts of PFAS and how we wanted to contribute to solve this problem by developing a PFAS detection device for ski wax. This blog article is still under the process of being published, as of now. \nWe also contacted Titan, a digital newspaper at the department of science and technology at the university of Oslo, to hear if they were interested in interviewing us, which they were. In the interview with Titan, we talked about our project and motivation behind it. This interview will be published right before the Jamboree, thus they wanted to follow our journey and the development of our detection device up to that point. \n\n### Future directions:\n\nWe hope to be able to get more media coverage in the near future to promote our project and convey the message regarding PFAS to an even larger audience. We have the ambition to reach out to podcasts, and other papers and magazines. By then, our goal is to have worked more on the project and have new and interesting results to present. \n\n### Katrine Borgå (ecotoxicologist at UiO):\n\nQuite early on our project we arranged a meeting with Katrine Borgå. At this time, we just learned about the negative impacts of PFAS, which frequently is used in ski wax. We had already decided to tackle this problem by specifically developing a detection system for PFAS containing ski wax. As we were in the starting phase of the project, wanted to talk to an expert within this field to get to know these chemicals better to know where we potentially could attack this problem. \nKatrine Borgå is an ecotoxicologist at the University of Oslo. Her research focuses on understanding mechanisms and processes within contamination distribution and accumulation in the environment, including PFAS. During this meeting Katrine presented several results from research on PFAS and negative impacts in health and accumulation in the environment, both her own and others results. We also had a great discussion on how these chemicals are labeled and used in the industry, to prevent the national regulation we have on these kinds of chemicals in Norway. \nHer presentation and our discussion at the end of the meeting showed us a bigger picture of the problem. Her research and results from other research underpin the urgent need of better regulation of these chemicals. We learned a lot about the negative impacts of the human health and the environment from this that we were not aware of. We therefore realized after this meeting that this problem is not widely spoken about enough in the public and decided that we wanted to change that by making a big part of our project being spreading awareness regarding this chemical. This meeting was quite valuable for the starting phase of our project.\n\n### Skiforbundet (the Norwegian skiing association):\n\nSince we looked into the idea of developing a detection mechanism for PFAS in ski wax, we wanted to hear some thoughts regarding this idea and the feasibility of the project from the skiing community. We therefore invited the Norwegian skiing association (Skiforbundet) for a meeting. Two specialists within ski wax appliers and a technical sport chemist from Skiforbundet joined us for the meeting. \nDuring this meeting we got a better understanding of the ski wax situation in the skiing community and why no actions have been taken since the ban was officially announced in 2019 by FIS (The international skiing association – Fedération Internationale de Ski). We learned more about the existing detection mechanisms that they have tried out and looked into, which have not satisfied their needs. From this meeting we therefore got a grasp of what to focus on when designing a detection mechanism specifically for use during skiing competitions. \nOne of the members from Skiforbundet stated that there will be no innovation without the development of a functional detection device for ski wax during the meeting. This made us realize the important impact of such a detection device, which will put the pressure on ski wax manufactures to find new ways to develop new and more environmentally friendly products in the future. \nThis meeting with Skiforbundet was a huge success. We were asked important questions that we had yet thought about and touched upon subjects that were relevant for us, which drove our project in the right direction. At the end of this meeting, we also got the contact information for relevant persons from SWIX, one of the largest skiing equipment manufacturers in Norway).\n\n# Wet lab results\n\nTo clone and express the desired dehalogenase gene for our project, we tried to isolate the gene from two different bacteria strains (Burkholderia cepacia and Pseudomonas aeruginosa) in parallell to see which would be most applicable for our project. \n\n## Dehalogenase from Burkholderia cepacia\n\n### Cloning of gene into expression vector\n\n**Goal:**\n\nCloning of gene isolated using PCR into the expression vector pBAD using Gibson assembly, and transformation of competent cells. \n\n**Results:**\n\nTo find the most optimal cloning conditions, we tried out different cloning paramters by changing: ratio of insert to vector, and incubation times. \nGene from *B.cepacia* and pBAD vector was assembled by Gibson assembly. *Escherichia coli* chemically comptetent cells (DH5α) was transformed with the assembly. \nAfter successful transformation, colony PCR was performed to check if the colonies contained the plasmid with the insert. \n\n### Optimalization of cloning paramters\n\n**Ratio of insert to vector: 1:1**\n**Incubation: 50 °C for 15 minutes.**\n \n\n|Figure 1: Agar plate with colonies. Insert to vector ratio was 1:1. Incubation time for Gibson assembly was 15 min at 50 °C.\nNumber of colonies: 23.\n\n**Ratio of insert to vector: 3:1**\n\n**Incubation: 50 °C for 60 minutes.**\n \n\n|Figure 2: Agar plate with colonies. Insert to vector ratio was 3:1. Incubation time for Gibson assembly was 1 hour at 50 °C.\nNumber of colonies: 1\n\n**Ratio of insert to vector: 6:1**\n\n**Incubation: 50 °C for 15 minutes.**\n \n\n|Figure 3: Agar plate with colonies. Insert to vector ratio was 6:1. Incubation time for Gibson assembly was 15 min at 50 °C.\nNumber of colonies: 10\n\n**Ratio of insert to vector: 6:1**\n\n**Incubation: 50 °C for 60 minutes.**\n \n\n|Figure 4: Agar plate with colonies. Insert to vector ratio was 6:1. Incubation time for Gibson assembly was 1 hour at 50 °C.\nNumber of colonies: 0\nThe cloning conditions that yielded most colonies was Ratio: 1:1 and 15 min incubation at 50 °C. \nColony PCR of colonies from the cloning and transformation\n \n|Figure 5: Gel image after gel electrophoresis of product from the colony PCR of \ncolonies from plate with ratio 1:1 of insert and vector.\n\nThe primers used for the colony PCR are designed to amplify the insert if it is inserted into the expression vector. The gene of interest has a size of ~900 bp. The result from the colony PCR shows that bands at right below 1000 bp for all of the colonies, meaning that all of the colonies from the PCR contained the vector with the insert. \nTo verify that the insert had the correct DNA sequence, miniprep was performed to extract the vector-insert construct from the cells, and it was sent for sequencing. \nThe sequencing result came back showing that indeed the DNA sequence of the gene was identical with the DNA sequence of the gene of interest. \n\n### Expression of protein from Burkholderia sp.\n\n**Goal:**\n\nProve that the Dehalogenase protein can be overexpressed in *E. coli* BL21 competent cells by the addition of L-arabinose, thus making it possible to proceed with protein purification.\n\n**Results:**\n\nBL21 *E. coli* competent cells were transformed with the insert-vector construct that we confirmed contained the gene encoding a Dehalogenase. The expression was induced by adding L-arabinose.\nThe SDS-PAGE shows a strong band at ~35 kDa which is the size of the Dehalogenase protein. The overexpression of the Dehalogenase protein was thus successful. \nFrom this experiment it was also shown that the overexpressed protein was in the insoluble fraction. This means that the folding of the protein *in vivo* was incorrect, resulting in the formation of inclusion bodies. This result would dictate how the subsequent purification experiment was performed. \n \n\n|Figure 6: SDS-PAGE gel with a strong band at ~35 kDA.\n\n### Purification of protein \n\nThe dehalogenase from Burkholderia sp was purified using immobilized metal affinity chromatography (IMAC) with nickel NTA (Ni-NTA) resin \n\n |Figure 7: SDS-PAGE gel of purification attempt of the dehalogenase using nickel-NTA beads. \nThe purification attempt with the nickel NTA resin was not satisfactory. Improvements could be made using a nickel affinity column mounted on an Äkta system.\n\n### Protein refolding\n\nTo find different refolding conditions to try, we used as a guideline the iFOLDTM Protein Refolding System 1 from Novagen®. This system contains 92 different buffers with unique combinations of salts concentrations and pH. We picked out a few alternatives that were available for us in the lab. We used a Tris-based buffer and we decided to use different salt concentrations as shown below. \n \n1) NaCl variations in concentration with Tris-buffer. \n * 0 mM NaCl\n * 150 mM NaCl (this is similar to what would be the natural concentration in a cell)\n * 350 mM NaCl \n2) Other variations\n * Glycerol (10%)\n * 500 mM Arginine \nThese are conditions are based on refolding conditions that have been shown to work experimentally.\n \n\n|Figure 8: Silver stained SDS-PAGE loaded with protein in different refolding buffers.\nSince the protein sample was not very pure, some contaminants were also refolded. Based on this gel, the better refolding buffer was Tris + 10 % Glycerol.\n\n## Pseudomonas aeruginosa\n\n### Cloning of gene encoding dehalogenase from Pseudomonas aeruginosa\n\n**Goal:**\n\nClone gene from genomic DNA of P. aeruginosa isolated and amplified by PCR into the expression vector pBAD using Gibson assembly, and transformation of competent cells.\n\n**Result:**\n\nThe PCR products were loaded onto an agarose gel and separated by size using gel electrophoresis. We expect the gene to be around 900 bp, and in the gel the band is right below 1000 bp, indicating the correct band size. The pBAD vector has a size of 3906 bp. In this image, the vector appears to be right above 3000 bp.\n |Figure 9: PCR products on agarose gel after gel electrophoresis. The same PCR product was loaded in both of the wells annotated Insert. The same PCR product was also loaded in the wells annotated pBAD vector. \n\n |Figure 9: PCR products on agarose gel after gel electrophoresis. The same PCR product was loaded in both of the wells annotated Insert. The same PCR product was also loaded in the wells annotated pBAD vector. \nSince the gel showed bands at the correct size, the next step was cloning of insert into the vector. \nThe gene isloated by PCR was cloned into the pBAD vector by Gibson assembly, and competent **E. coli** DH5α cells were transformed with the assembly product. \nThe transformation yielded two colonies, and both were analysed by colony PCR to verify the presence of the gene of insert. \n \n\n|Figure 10: Two colonies were formed after transformation with insert-pBAD construct. \n\n |Figure 11: Gel image of PCR products from colony PCR.\nBoth colonies from the cloning attempt were checked for the insert by performing colony PCR. From the gel image of the PCR result, only colony 2 seems to have the insert. Miniprep was performed of both cultured colonies, and both plasmids were sent for sequencing to verify the presence of the gene with correct DNA sequence. The sequencing results from colony 1 turned out to be the correct sequence. Although the insert did not appear on the gel after colony PCR, the concentration may have been too low for the PCR to sucessfully amplify the insert. \nThe sequence result was run through BLASTX to find the protein the DNA sequence encoded. There was a match with 100.0 % sequence identity for alpha/beta hydrolase which is the protein family of the Dehalogenase. Data from the Protein Data Bank showed that the mass of the protein was ~35 kDa. With this information, we proceeded with protein expression.\n\n### Expression of dehalogenase protein from P. aeruginosa\n\n*Goal:* Overepxress the Dehalogenase protein in E. coli BL21 competent cells by the addition of L-arabinose and investigate if the dehalogenase protein from P. aeruginosa would be in the soluble or insoluble fraction thus making the purification step less complicated. \n*Result:* The SDS-PAGE showed a strong band at ~35 kDa for the samples where L-arabinose was added, but not in the sampels where arabinose was not present, indicating that the overexpression of the protein was successful. On the other hand, also the dehalogenase from P. aeruginosa was in the insoluble fraction. Thus the subsequent purifictation would be as complicated and would involve a refolding step as for the protein from B. cepacia. \n\n |Figure 12: SDS-PAGE showing the samples from the overexpression experiment together with an uninduced control. \n\n### Purification of protein from Pseudo\n\nThe Dehalogenase from P. aeruginosa was purified with a nickel affinity column mounted on an Äkta system and eluted with 250 mM imidazole.\n \n\n|Figure 13: Purification of Dehalogenase from *P. aeruginosa.*\nLooking at the SDS-PAGE gel from the purification, there were still some contaminations in the fractions. To improve this, the imidazole gradient could have been halted at 50 % elution buffer to remove more of the impurities. \n\n### Protein refolding\n\nTo find different refolding conditions to try, we used as a guideline the iFOLDTM Protein Refolding System 1 from Novagen®. This system contains 92 different buffers with unique combinations of salts concentrations and pH. We picked out a few alternatives that were available for us in the lab. We used a Tris-based buffer and we decided to use different salt concentrations as shown below. \n 1) NaCl variations in concentration with Tris-buffer. \n * 0 mM NaCl\n * 150 mM NaCl (this is similar to what would be the natural concentration in a cell)\n * 350 mM NaCl \n2) Other variations\n * Glycerol (10%)\n * 500 mM Arginine \nThese are conditions are based on refolding conditions that have been shown to work experimentally.\n \n\n|Figure 14: Silver stained SDS-PAGE loaded with protein in different refolding buffers.\nFor the dehalogenase from P. aeruginosa it was the folding conditions with Tris + 350 mM NaCl and Tris + 500 mM Arginine that gave the best refolding results. The band is at size ~35 kDa. \n\n### Testing the protein with PFOA\n\nBefore we tested the enzymatic defluorination we wanted to verify that the tests worked. The test is based on a color change when fluoride ions are present in a solution. The test only works at low pH levels; therefore, an acid must be added to the sample before application. \n\n|Figure 15: Different concentrations of NaF were tested using the colorimetric fluoride test compared with illustrations of how different concentrations of F- should appear on the patches.\nAfter we verified that the fluoride test worked, we moved on to test if free fluoride ions could be detected when the dehalogenase was exposed to PFOA. \n\nWe concentrated the dehalogenase from *P. Aeruginosa* after the purification and exchanged the refolding buffer with a buffer of 100 mM Tris, pH 9.5. \n\nPFOA was diluted in the same buffer as the protein. Two different concentrations of PFOA were tested: 20 mM and 10 mM. In addition, we had a negative control with only the dehalogenase, and another negative control with 20 mM of PFOA without the protein. \n20 mM NaF served as a positive control. \n\nThe protein concentration was quantitatively estimated using a BCA assay with a Bovine Serum Albumin standard of known concentrations and estimated to be 27.5 µg/mL. \n\nThe test discs are incubated with sample for 5-7 minutes to allow the disc to be saturated. \n\n \n|Figure 16: Samples of PFOA + protein with different concentrations of PFOA, negative and positive controls. The bottom row is the color standards provided by the manufacturer.\n \n\nThe size of the bleached area corresponds to the total fluoride concentration. Looking at Figure 6, there is a clear difference between the negative controls where no bleaching has taken place, and the discs with sample containing enzyme together with PFOA where there is a distinct bleached area. According to the test protocol, this indicates that there might be fluoride ions in the sample with enzyme and PFOA. ',qn='# Parts \n\n \n\n \n\nWe worked with two parts for this year’s project: pBAD expression vector with a dehalogenase from Burkholderia sp. and pBAD expression vector with a putative epoxide hydrolase from Pseudomonas aeruginosa. \n\n \n\n| **Part number** | **Short description** | \n|--|--| \n| ["BBa_K3709000"](/wiki/index.php?title=Special:Redirect/file/T--UiOslo_Norway--http://parts.igem.org/Part:BBa_K3709000) | Fluoroacetate Dehalogenase from Burkholderia sp. FA1| \n| ["BBa_K3709002"](/wiki/index.php?title=Special:Redirect/file/T--UiOslo_Norway--http://parts.igem.org/Part:BBa_K3709002) | Putative epoxide hydrolase from Pseudomonas aeruginosa | \n\n \n\n## BBa_K3709000: Fluoroacetate Dehalogenase \n\n \n\nThis gene was synthesized as a gBlock by IDTDNA containing a His tag and TEV cleavage site, and is codon optimized for expression in Escherichia coli. The gene is cloned into pBAD vector containing the araBAD promoter, and expression is induced with L-arabinose. \n\nFigure 1: Plasmid map of pBAD expression vector containing the Dehalogenase sequence as a gBlock from IDTDNA. \n\n\n\n \n\n## BBa_K3709002: Putative epoxide hydrolase \n\n \n\nThe putative epoxide hydrolase from *P. aeruginosa* was chosen due to its homology with the fluoroacetate dehalogenase from Burkholderia sp. The gene was isolated from genomic DNA of P. aeruginosa and cloned into the pBAD expression vector. The primers were designed to include 6xHis-tag and TEV cleavage site from the gBlock designed for the dehalogenase from Burkholderia sp. \n\n \n\n \n\nFigure 2: Plasmid map of pBAD expression vector containing the gene for epoxide hydrolase from \nP. aeruginosa. \n\n \n\n \n\n ',Hn="# General Laboratory Protocols \n\n \n\n## Chemically Competent E. coli cells \n\nTwo different strains of competent E. Coli were used: \n\n* BL21 E.coli cells for protein expression \n\n* DH5α cells for cloning. \n\n \n\n## Dpnl digestion \n\nAdd 1 uL of DpnI enzyme in a tube with the PCR-product. Incubate for 1h at 37 °C \n\n \n\n## Bacterial Transformation \n\n1. Mix 5µl of DNA with 70µl of chemically competent cells \n\n2. Incubate on ice for 15 min \n\n3. Heat shock for 45 sec at 42oC \n\n4. Place on ice for 2 min \n\n5. Rescue with addition of 700 µl SOC (LB can work too) \n\n6. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. \n\n7. Warm selection plates to 37°C. \n\n8. Spin down max speed 15sec, decant most of the supernatant and Spread 50–100 µl of the cells and ligation mixture onto the AMP plates. \n\n9. Incubate overnight at 37°C \n\n10. Check plates for colonies. The plates can be stored at 4C for a few days, if they are wrapped in parafilm. \n\n \n\n \n\n## Plasmid Miniprep \n\nMiniprep with Monarch® DNA Gel Extraction Kit (#T1020S) from New England BioLabs®, as described in the kit’s protocol. \n\n \n\n \n\n## Agarose Gel \n\n0.8% agarose in TAE and 10% GelRed® Nucleic Acid Stain. \n\n \n\n \n\n## Agarose Gel Electrophoresis \n\nRan gel electrophoresis at 90 V for 30 minutes. \n\n \n\n \n\n## Gibson Assembly \n\nUse this equation to calculate the needed amount of insert and vector: \n\npmols = (weight in ng) x 1000 / (base pairs x 650 Da) \n\n \nIt is recommended a total of 0.02-0.5 pmols of DNA fragments for assembly of 1-2 fragments into a vector. \n\n \n\n1. Set up the following reaction on ice: \n\nThe calculated amount of the insert and the vector \nSame amount as the added DNA of Gibson Assembly Master Mix (2X) \nFill up to a total volume of 20 uL with ddH2O \n\n2. Incubate in a thermocycler at 50C for 15 min. After incubation, the samples can be stored on ice or at –20C for subsequent transformation. \n\n \n\n| Components | Volume (50 𝜇l in total)| \n|--|--| \n| 5X GC buffer | 10 𝜇l | \n| 10 mM dNTPs | 1 𝜇l | \n| 10 𝜇M Forward primer | 2.5 𝜇l | \n| 10 𝜇M Reverse primer | 2.5 𝜇l | \n| Template DNA | 0.5 𝜇l | \n| Phusion polymerase | 0.5 𝜇l | \n| H2O | 33 𝜇l | \n\n \n\n \n\n| Step | Temp | Time | \n|--|--|--| \n| Initial denaturation | 98 ℃ | 30 s | \n| 28 cycles | 98 ℃ | 10 s | \n| 28 cycles | 68 ℃ |30 s | \n| 28 cycles | 72 ℃ |15 s | \n| Final extension | 72 ℃ | 7 min | \n| Hold | 12 ℃ | ∞ | \n\n \n\n## PCR clean-up \n\nPCR clean-up with MinElute® PCR Purification Kit (Cat. No. 28006) from Qiagen®, as described in the kit’s protocol. \n\n \n\n## Colony PCR \n\n1. Incoulate PCR reaction mix with colony from cloning. \n\n2. Also streak a plate and inoculate 5 mL LB + antibiotics to do miniprep for sequencing. \n\n3. Run colony PCR protocol as illustrated in figure 1. \n\n4. Load on an agarose gel and run gel electrophoresis to investigate if the colony contained the vector with insert. \n\n \n\n \n\n![Figure 1. PCR program.] (PCR_Program.png) \n\n \n\n## SDS-Page \n\nGels: Novex wedge well 4-20 % Tris-Glycine gel, 15 wells. \n\nRun at 225 V for 40 minutes. \n\n \n\n \n\n## SDS staining solution \n\n400 mL H20 \n\n500 mL EtOH \n\n100 mL Acetic acid \n\n1.25 g Coomassie \n\n \n\n \n\n## SDS running buffer \n\nMeasure out the needed amount of the solutions as described below and mix in a large flask. This solution can be stored for a longer period in room temperature. \n\n100 mL 10x Tris-glycerin buffer \n10 mL 10% SDS \n890 mL ddH2O \n\n \n\n \n\n## Glycerol Stock \n\n1. Streak plate containing antibiotics with bacteria from glycerol stock. Incubate at 37 °C overnight. \n\n2. Pick one colony and inoculate 5 mL of LB + 50ug/ml antibiotics. \n\n3. Grow at 37C until OD ~0.4-0.6. \n\n4. Transfer the needed amount of glycerol and bacterial culture to a cryotube to make a final concentration of 20% glycerol in a total volume of 2 mL (pre1:1 ratio of glycerol and bacterial culture). Carefully mix and immediately put on ice. \n\n5. Store cryotube to –80 °C freezer. \n\nWhen the glycerol stock will be used again, take a small inoculating stick and dip it in the stock before streaking it on a plate. Have the glycerol on ice through the whole process and work quick so the glycerol stock does not thaw. \n\n \n\n \n\n## References \n\nAll figures on this page have been created in Biorender.com. \n\n \n\n ",Xn="# Protein purification under dentaturing conditions for medium level expression \n\n \n\n## For medium level of expression: \n\n(you can ignore this step as you will start from the frozen pellet anyway ) Harvest cells from a 50 ml culture by centrifugation (5,000 x g for 10 min) \n\nResuspendpellet in 2 ml Denaturing Lysis Buffer \n\n \n\nStir cells for 15 min (up to 60 min) at room temperature [lysis is complete when solution becomes translucent] If necessary, sonicate with 3x 30sec high intensity pulses (this will be needed) and 2 mM MgSO4, 0.01 mg/mL DNAse and 0.1 mg/mL lysozyme were added. The solution was incubated at room temperature for 15 min. \n\n \n\nCentrifuge lysate at 10,000 x g for 20 min at room temperature to pellet cellular debris → Keep supernatant \n\n(In case SDS-PAGE analysis is needed GuHCl does not mix with SDS, instead: take 5 µl supernatant, make 100 µl with water, add 100 µl of 10 % TCA, incubate 20 min on ice, centrifuge max speed 15 min. Wash pellet with 100 µl 100% ethanol and centrifuge at max speed for 15 min, remove ethanol and be careful not to touch pellet. The pellet will not be visible to the naked eye at this point, so be careful to remove ethanol at opposite side of the pellet. Therefore you need to be careful of what side is up and down in the centrifuge. After removing ethanol resuspend sample in 1x SDS-loading buffer [100 µl] , boil sample at 95oC for 10 min) \n\n \n\n Prepare the Ni-NTA slurry by pipetting 250 µl of the Ni-NTA agarose into a centrifugation tube. Gently pellet it by low-speed centrifugation (700 x g for 2 min). Gently aspirate and discard the supernatant. \n\n* 250µl of 50% NiNTA slurry is enough for 15 mg of protein (bed volume = 125µl) \n\n \n\nAdd 6 resin-bed volumes (i.e. 1.5 ml) of distilled water and resuspend by inverting. Gently pellet it by low-speed centrifugation (700 x g for 2 min). Gently aspirate and discard the supernatant. \n\n \n\nAdd2 resin-bed volumes (i.e. 250 µl) of Denaturing Equilibration Buffer and mix until the resin is fully resuspended. Gently pellet it by low-speed centrifugation (700 x g for 2 min). Gently aspirate and discard the supernatant. \n\n \n\nAdd the 2 ml lysate to the prepared Ni-NTA resin and mix gently by shaking ~200 rpm for 30 min at room temperature \n\n \n\nCentrifuge tube (700 x g for 2 min). Gently aspirate and discard the supernatant. (if required keep some of supernatant for SDS-PAGE analysis) \n\n \n\nWash the resin with 2 resin-bed volumes (i.e. 250 µl) of Denaturing Wash Buffer and mix until the resin is fully resuspended. Gently pellet it by low-speed centrifugation (700 x g for 2 min). Gently aspirate and discard the supernatant. (if required keep some of supernatant for SDS-PAGE analysis) \n\n \n\n(Repeat wash step and monitor supernatant by measuring absorbance at 280nm until baseline is reached) - in our case this step was skipped \n\n \n\nElute bound His-tagged protein using 1 resin-bed volume (i.e. 150 µl) of Denaturing Elution Buffer and mix until the resin is fully resuspended. Gently pellet it by low-speed centrifugation (700 x g for 2 min). Gently aspirate and keep the supernatant. This should be repeated at least 3 times, 4 times is what we did just to be sure, resulting in 4 samples from supernatant. \n\n \n\nRepeat elution step 2x and monitor supernatant by measuring absorbance at 280nm or perform Bradford/BCA assay \n\n \n\nRefolding: \n\nRapid Dilution: Dilute your protein in high urea into a buffer without urea. The volume of the final buffer should be large enough to make a low final concentration of Urea (I usually go for less than 0.5M). Do 20x dilution. \n\nOR Dialysis: This is similar to rapid dilution, but will happen over a longer time period. Dialyze your protein into a final buffer without urea. A good rule of thumb is to do a 10X buffer exchange at least 2 times. \n\n \n\n \n\nFigure 1: Work flow of protein purification \n\n \n\n ",Zn="# Medium-scale protein expression for purification \n\n \n\n(Modified from 2018 [**UiOslo iGEM team CanndiTect**](https://2018.igem.org/Team:UiOslo_Norway) ) \n\n \n\n* Transform BL21 (New England BioLabs) cells with expression plasmid. \n\n* Make overnight cultures of single colonies grown in LB media supplemented with maintenance antibiotics. Incubate at 37 °C 200 rpm overnight. \n\n* Dilute the overnight cultures 1000-fold in 150 mL fresh LB media with antibiotics and incubate at 37 °C, 200 rpm, until OD600 ~0.4-0.6. \n\n* Induce with arabinose at final concentration of 0.2 %. \n\n* Keep one tube aside as negative control, 5 mL in 15 mL falcon tube. \n\n* Incubate at 37 °C, 200 rpm, for a minimum of 4 hours. \n\n* Normalize the OD in the induced and non-induced samples. \n\n* Isolate cells by centrifugation at 5000 xg for 10 min. Keep the cell pellet, discard the supernatant. \n\n* Use pellet for Protein purification protocol Link til purification protocol (His-Tag Purification denaturing conditions) ",Vn="# Protein re-folding parameters \n\n \n\n**Set ups, each with 5 µL of protein in a 1:20 dilution:** \n\n \n\n**Buffer** \n\n \n\n* Denaturing Elution buffer [95 µL] (Control) \n\n \n\n* Tris + 0 M NaCl [95 µL] \n\n \n\n* Tris + 0.150 M NaCl [95 µL] \n\n \n\n* Tris + 0.350 M NaCl [95 µL] \n\n \n\n* Tris + 0.5 M Arginine [95 µL] \n\n \n\n* Tris + 10% Glycerol [95 µL] \n\n \n\n**1.** The Eppendorf tubes [1.5 mL] is used when adding. The protein + buffer mixture needs to be vortexed immediately after the protein is added. Then left out in room temperature for minimum 30 min. \n\n \n\n \n\n**2.** Centrifuge at full speed for 10 min. \n\n \n\n \n\n**3.** 10 µL or 20 µL is taken from the supernatant and mixed with 4x or 6x SDS sample buffer. \n\n \n\n \n\n**4.** Boil it for 5 min and load onto SDS-PAGE gel. \n\n \n\n \n\n**5.** Run gel at 225 V for 40 min. ",_n="# Larger-scale protein refolding \n\n \n\n* To refold the protein, the denaturing urea buffer must be diluted 1:20 in an appropriate refolding buffer. \n\n* Concentrate the protein by centrifugation in a spin column with an appropriate molecular weight cut-off. \n\n* Monitor the absorbance of the supernatant at 280 nm and stop centrifuging when the absorbance (280) has reached peak. \n\n* Measure the protein concentration using Pierce™ BCA Protein Assay Kit (Thermo FischerTM). \n\n",$n="# Silver staining \n\n \n\n### Protocol as described in “A simple modification of Blum’s silver stain method allows for 30 minute detection of proteins in polyacrylamide gels” by **Michael V. Nesternko**, **Micheal Tilley** and **Steve J. Upton** (Biochemical and biophysical methods, 1993) with a few modifications. \n\n \n\n**Important notes: All the steps were in a glass container on a shaker at room temperature.** \n\n \n\n**Stock solutions**: \n\n50% acetone in ddH2O \n\n50% TCA in ddH2O \n\n20% AgNO3 in ddH2O \n\n10% Na2S2O3 H2O in ddH2O \n\n \n\n**1.** When the SDS-PAGE is done running, carefully remove and place the gel in a glass container that are large enough to hold the whole gel. Rinse the gel breifly in ddH2O, discard the water and place the container on a shaker. \n\n \n\n**2.** Optional: prepare all of the needed solutions for the next steps. From here on, the solutions described for each step are mixed in a falcon tube and added to the container with the gel. Incubate as described and discard the solution before the next solution is added. \n\n \n\n**Fixation**: 30 mL acetone stock, 0.75 mL TCA stock and 12.5 uL. Incubate for 5 min. \n\n \n\n**Rinse**: ddH2O (fill up to cover the gel). Incubate for 5 sec. Repeat this step 3 times. \n\n \n\n**Wash**: ddH2O (fill up to cover the gel). Incubate for 5 min. \n\n \n\n**Rinse**: ddH2O (fill up to cover the gel). Incubate for 5 sec. Repeat this step 3 times. \n\n \n\n**Pretreat**: 30 mL acetone stock. Incubate for 5 min. \n\n \n\n**Pretreat**: 50 uL Na2S2O3 H2O stock in 30 mL ddH2O. Incubate 1 min. \n\n \n\n**Rinse**: ddH2O (fill up to cover the gel). Incubate for 5 sec. Repeat this step 3 times. \n\n \n\n**Impregnate**: 0.4 mL AgNO3 stock, 0.3 mL 37% HCHO, 30 mL ddH2O. \n\n \n\n**Rinse**: ddH2O (fill up to cover the gel). Incubate for 5 sec. Repeat this step 2 times. \n\n \n\n**Develop**: 0.6 g Na2CO3, 12.5 uL 37% HCHO, 12.5 uL Na2S2O3 H2O stock, 30 mL ddH2O. Incubate until the desired staining is achieved, then quickly discard the develop solution and pour in the stop solution as described in the next step. For optimal staining, start discarding the develop-solution a few seconds before the desired staining is obtained, thus the staining still continue for a few seconds after the stop-solution has been added. \n\n \n\n**Stop**: 1% glacial acetic acid in ddH2O ",et="# Small-scale protein expression \n\n \n\n### Edited from [**CandiTect UiO**](https://2018.igem.org/Team:UiOslo_Norway) (2018) iGEM team. \n\n \n\n* Transform BL21 (New England BioLabs) cells with expression plasmid. \n\n* Make overnight cultures of single colonies grown in LB media supplemented with maintenance antibiotics. Incubate at 37 °C 200 rpm overnight. \n\n* Dilute the overnight cultures 1000-fold in fresh LB media with antibiotics and incubate at 37 °C, 200 rpm, until OD600 ~0.4-0.6. \n\n* Induce with arabinose at final concentration of 0.2 %. \n\n* Keep one tube aside as negative control. \n\n* Incubate at 37 °C, 200 rpm, for a minimum of 4 hours. \n\n* Normalize the OD in the induced and non-induced samples. \n\n* Isolate cells by centrifugation at 5000 xg for 10 min. Keep the cell pellet, discard the supernatant. \n\n* Resuspend pellet in appropriate amount of LB to make a concentration of 1 g/ml cells: \n\n * OD600 = 1 => 1.7 g/mL of cells. \n\n * Amount of cells in sample = Volume of sample * OD of sample. \n\n * Grams of cells in sample = amount of cells (OD) * 1.7 g/mL \n\n * Volume to resuspend pellet in = grams of cells/1.7 g/mL \n \n\nTake 1 g of cells (1 mL) and transfere it to a 1.5 mL eppendorf tube. \n\n* Centrifuge at 8K xg for 2 min and discard supernatant. \n\n* Resuspend pellet in 150 ul B-PER. \n\n* Centrifuge again at 8K xg for 2 min and discard supernatant. \n\n* Add another 1 mL of cells to the eppendorf tube. Spin down at 8K xg for 2 min. Discard supernatant. \n\n* Resuspend pellet in 150 ul B-PER + 0.1 mg/mL lysozome, 0.01 mg/ml DNAse, 10 med mer MgCl2 and 1mM PMSF. \n\n* Leave for 15 min at room temperature. \n\n* Centrifuge at 8K xg for 2 min. Keep supernatant as soluble fraction. \n\n* Add another 150 ul B-PER. Resuspend the pellet. This is the insoluble fraction. \n\n* To 37.5 ul sample add 12.5 uL 4X Laemmli sample loading buffer containing \n2 mM dithiothretiol (DTT). \n\n* Vortex samples. \n\n* Incubate samples at 95 °C for 10 min. \n\n* Load 12 ul of each soluble fraction and 6 ul of each insoluble fraction onto a SDS-PAGE gel. Performe gel electrophoresis at 225 V for 40 min to separate samples based on size in 1X SDS running buffer. \n\n* Stain gel with Coomassie Blue reagent for 30 min. Then destain the gel for 30 min. Then leave the gel in water overnight before imaging the gel. ",nt="# Proof of concept  \n\n  \n\nThe ultimate goal of our project was to be able to detect PFAS in ski wax. As a proof of concept, we have used a colorimetric based test to indicate the presence of free fluoride ions in a solution of perfluorooctanoic acid (PFOA) after incubation with a Dehalogenase enzyme from *Pseudomonas aeruginosa*.    \n\n \n\nThe team worked with two proteins from two different strains of bacteria: *Burkholderia sp*. and *Pseudomonas aeruginosa*. We managed to both express and purify the proteins in *Escherichia coli*. \n\n\n \n|Figure 1: SDS-PAGE gel with a strong band at ~35 kDA. \n\n \n\n\n \n|Figure 2: SDS-PAGE showing the samples from the overexpression experiment together with an uninduced control. \n\n \n\nThe size of both the proteins was around 35 kDa, Figure 1 and Figure 2 shows that we have a band at ~35 kDa in the samples where expression was induced with arabinose. \n\n \n\nThe protein was in the insoluble fraction, most likely in inclusion bodies. To purify the proteins, we had to denature the proteins in high concentrations of urea, and then attempt to refold them to their native conformation by diluting the urea 1:20. This step requires a lot of trial and error to find buffer conditions that allows the protein to refold. We tried different buffers with different additives, resulting in successful refolding of the proteins of interest. If the protein has refolded, it would be in the supernatant, and therefore the sample was centrifuged, and the supernatant was loaded onto an SDS-PAGE gel. \n\n \n\n \n\n\n \n|Figure 3: Silver stained SDS-PAGE loaded with protein from *Burkholderia sp.* in different refolding buffers. \n\n \n|Figure 4: Silver stained SDS-PAGE loaded with protein in different refolding buffers. \n\n \n\nFigures 3 and 4 show the results of the refolding attempt on the dehalogenase from *Burkholderia* sp. and *P. Aeruginosa* respectively. Both of the silver-stained gels show that a band has appeared at ~35 kDa for several of the refolding buffers. This indicates that the protein of interest was present in the supernatant and thus has been refolded. \n\nThe gel loaded with protein from *Burkholderia* sp. show two distinct bands in each sample due to unsatisfactory purification. \n\n \n\nNow that functional refolding buffers had been identified for the dehalogenases from both of the organisms, we could proceed with the actual testing on PFOA. \n\n \n\nTo test for free fluoride ions in PFOA, we obtained a Fluoride test from Macherey-Nagel GmbH & Co. KG. This is a colorimetric test where the concentration range of F- can be determined based on color change. The test consists of pink test discs that are bleached by fluoride-containing hydrochloric acid solutions. To apply the sample, a pin hole is poked in the middle of the disc, allowing the sample to draw into pinhole and saturate the disk where the reaction takes place. Hydrochloric acid is added to the sample before applying the sample onto the test disc. \n\n \n\n## Test of colorimetric fluoride test  \n\n \n\n \n|Figure 5: Different concentrations of NaF were tested using the colorimetric fluoride test compared with illustrations of how different concentrations of F- should appear on the patches.\n \nBefore we tested the enzymatic defluorination we wanted to verify that the tests worked by testing with different concentrations of NaF (Figure 5). \n\nComparing the color to the standard, the result was accurate and reflected the concentration of the NaF sample we tested with. \n\n \n\n## Testing the protein with PFOA \n\n \n\nAfter we verified that the fluoride test worked, we moved on to test if free fluoride ions could be detected when the dehalogenase was exposed to PFOA. \n\nWe concentrated the dehalogenase from *P. Aeruginosa* after the purification and exchanged the refolding buffer with a buffer of 100 mM Tris, pH 9.5. \n\nPFOA was diluted in the same buffer as the protein. Two different concentrations of PFOA were tested: 20 mM and 10 mM. In addition, we had a negative control with only the dehalogenase, and another negative control with 20 mM of PFOA without the protein. \n\n20 mM NaF served as a positive control. \n\nThe protein concentration was estimated to be 27.5 µg/mL. \n\nThe protein concentration was quantitatively estimated using a BCA assay with a Bovine Serum Albumin standard of known concentrations and estimated to be 27.5 µg/mL. \n\n \n\n \n\nThe test discs are incubated with sample for 5-7 minutes to allow the disc to be saturated. \n\n \n\n \n|Figure 6: Samples of PFOA + protein with different concentrations of PFOA negative and positive controls. The bottom row is the color standards provided by the manufacturer.\n\n \n\nThe size of the bleached area corresponds to the total fluoride concentration. Looking at Figure 6, there is a clear difference between the negative controls where no bleaching has taken place, and the discs with sample containing enzyme together with PFOA where there is a distinct bleached area. According to the test protocol, this indicates that there might be fluoride ions in the sample with enzyme and PFOA. \n\n \n\nThe test is made to be used on water-based samples. As PFOA is compound which contains a hydrophilic and hydrophobic part, we were worried that this would affect the test in relation to the saturation of the disk. \n\nThe controls saturated the entire disk and gave a clear negative result, exactly like when we tested it on deionized water and standard solution. If the PFOA influenced saturation of the disk, we would have seen this in the control containing only PFOA and buffer. \n\n \n\nTo verify this result, more extensive testing needs to be performed. By going back and optimizing the protein purification protocol to obtain a purer product, optimizing the refolding conditions, and hence increasing the protein yield. \n\nIt would also be interesting to look more into the concentration ratio of PFOA to protein to quantify at what ratio we have the optimal enzymatic activity. \n\n \n\n ",tt=function(){var e=this,n=e.$createElement,t=e._self._c||n;return t("div",{staticClass:"SponsAtt"},[t("Markdown",{attrs:{mdtext:e.attributions}}),t("Sponsors")],1)},ot=[],at="# Attributions \n\n \n\n## General support \n\n \n\nWe want to thank our support team who have assisted us as facilitators of the iGEM course at the University of Oslo [**BIOS4910IGEM – International Genetically Engineered Machine**](https://www.uio.no/studier/emner/matnat/ibv/BIOS4910IGEM) . Without their support with science, literature, meeting and workshop facilitations our team would not have reached this point of the competition. \n\n \n\n* Professor **Dirk Linke** \n\n \n\n* Postdoctoral Research Fellow **Athanasios Saragliadis** \n\n \n\n* Senior Academic Librarian **Kirsten Borse Haraldsen** \n\n \n\n* Professor **Finn-Eirik Johansen** \n\n \n\n* **The University of Oslo** for providing this as a course and the facilities, equipment, chemicals and fundings needed \n\n \n\n## Project support and advice \n\n \n\n* **Andrea Gasparini** Facilitated work-flow workshop \n\n \n\n* **NILU** (Norwegian Institute for Air Research) \n\n \n\n* Expert meeting with **Linda Hanssen** and **Vladimir Nikiforov** that provided us with knowledge about PFAS and the PFAS in ski wax. \n\n \n\n* Participants at our meeting with entrepreneurs and potential sponsors, here we received good feedback on both the project and further development. \n\n \n\n * **Inven2** a Norwegian company that helps the scientist to develop their ideas and patent it. \n\n \n\n * **The Life Science Cluster** a network for companies and organizations where life science is in focus. \n\n \n\n * **Sharelab** launchpad for start-ups, academics and companies that develop products and services using life science and biotechnology \n\n \n\n * **The Centre for Digital Life Norway** national centre for biotechnology research, education and innovation \n\n \n\n * **UiO:Life Science** our university, **UiO**, has started up a part of the university with a focus on Life Science. They are currently building a new building only for Life Science. \n\n \n\n## Fundraising help and advice \n\n \n\n* Professor **Finn-Eirik Johansen** for contacting and setting up a meeting with entrepreneurs and potential sponsors \n\n \n\n* iGEM Team [**NTNU Trondheim**](https://2021.igem.org/Team:NTNU-Trondheim) for providing us with inspiration for sponsoring templates \n\n \n\n## Lab support \n\n \n\n* Postdoctoral Research Fellow **Athanasios Saragliadis** \n\n \n\n* Head Engineer **Maria Chiara Di Luca** \n\n \n\n## Difficult technique support \n\n \n\n* Postdoctoral Research Fellow **Athanasios Saragliadis** with general support \n\n \n\n* Head Librarian for UiO Science Library **Simen Kjellin** for \n\n \n\n * Photgraphing and photo editing of portraits and team photo \n\n \n\n * Filming and editing of promotion video for our project \n\n \n\n * Assistance on presentation video \n\n \n\n* Doctoral Research Fellow **Ina Meuskens** with Silver Staining \n\n \n\n* Scientific Researcher **Simen Hermansen** for with protein purification \n\n \n\n## Wiki support \n\n \n\n* Academic Librarian **Dan Michael Heggø** for facilitating a wiki-workshop \n\n \n\n* iGEM Team [**NTNU Trondheim**](https://2021.igem.org/Team:NTNU-Trondheim) for sharing their experiences and feedback \n\n \n\n## Presentation coaching \n\n \n\n* Senior Academic Librarian **Tone Charlotte Gadmar** \n\n \n\n* Senior Academic Librarian **Karoline Moe** \n\n \n\n## Human Practices support \n\n \n\n* **Katrine Borgå** Expert meeting about PFAS, including her own and others research on the subject. Information about regulation and usage in industry was provided during this meeting \n\n \n\n* Collaboration with [**USAFA**](https://igem.org/Team.cgi) and [**FCB-UANL**](https://2021.igem.org/Team:FCB-UANL) on PFAS awareness campaign and panel \n\n \n\n* **Line S. Haug** from NIPH as guest lecture on PFAS awareness panel. Her experience and knowledge about PFAS, with focus on ski wax as a source to PFAS exposure and the negative health aspects was both insightful and informative. \n\n \n\n* Head of section for the Library of medicine and science **Jessica Lönn-Stensrud** for letting us present for students during her class \n\n \n\n* Meeting through **Skiforbundet** (Norwegian Ski Federation) \n\n \n\n * Expert meeting with **Stein Olav Snesrud** (Ski wax chief (professional ski wax applier) for Norway’s national cross-country team) \n\n \n\n * **The Norwegian Ski Federation** covers the national skiing disciplines of alpine, cross-country, freestyle, Nordic combined, ski jumping, and snowboarding \n\n \n\n * Two representatives from Olympiatoppen, **Felix Breitschadel** (Head of Equipment and Technology) and **Knut Nystad** (Project manager) \n\n \n\n * **Olympiatoppen** is an organisation under the Norwegian Olympic and Paralympic Committee and Confederation of Sports. Their responsibility is to train and develop athletes to compete in the olympic games and paralympics \n\n \n\n* **SWIX** A Norwegian manufacturing company of winter sports equipment, ski wax included. They answered our questions and gave us the possibility of providing us with samples of wax containing PFAS if needed. \n\n \n\n# Sponsors \n\n \n\n### We are incredibly grateful for our sponsors this year. Your support made us reach this point of the competition and we could not have made it without you. \n\n \n\n* **Akademika** for sponsoring us with 2 gift cards with the value of 250 NOK their bookshop at Blindern \n\n \n\n* **The Centre for Digital Life Norway** for sponsoring us with 50 000 NOK \n\n \n\n* **Eurofins** for sponsoring us with sequencing analyses \n\n \n\n* **IDT DNA** for sponsoring us with the gBlock of sequence encoding Fluoroacetate dehalogenase from *Burkholderia* sp. and primers. \n\n \n\n* **NILU** for sponsoring with PFOA sample and ski wax samples both with and without PFAS \n\n \n\n* **University of Oslo** for sponsoring us with funding, facilities, equipment and chemicals \n\n \n\n* **SNÖ** for sponsoring us with access to their facilities for filming our promotion video ",it=function(e){Object(h["a"])(t,e);var n=Object(d["a"])(t);function t(){var e;return Object(c["a"])(this,t),e=n.apply(this,arguments),e.attributions=at,e.sponsors=[{name:"NILU",imgUrl:"nilu.jpg"},{name:"Akademika",imgUrl:"akademika.png"},{name:"Center for Digital Life",imgUrl:"digital-life.gif"},{name:"Eurofins",imgUrl:"eurofins.png"},{name:"Snø",imgUrl:"sno.jpg"},{name:"University of Oslo",imgUrl:"uio.png"},{name:"IDT DNA",imgUrl:"idt.png"}],e}return t}(g["d"]);it=Object(u["a"])([Object(g["a"])({components:{Sponsors:De,Markdown:Ln}})],it);var rt=it,st=rt,ct=(t("66d5"),Object(F["a"])(st,tt,ot,!1,null,"23dc13cc",null)),lt=ct.exports,ht=function(){var e=this,n=e.$createElement,t=e._self._c||n;return t("div",{staticClass:"Team"},[t("b-card-group",{attrs:{columns:""}},e._l(e.membersA,(function(e,n){return t("TeamMember",{key:n,attrs:{name:e.name,text:e.text,imgUrl:e.imgUrl,discipline:e.discipline,funFact:e.funFact}})})),1),t("b-card-group",{attrs:{columns:""}},e._l(e.membersB,(function(e,n){return t("TeamMember",{key:n,attrs:{name:e.name,text:e.text,imgUrl:e.imgUrl,discipline:e.discipline,funFact:e.funFact}})})),1),t("b-card-group",{staticClass:"supervisors",attrs:{columns:""}},[t("b-card",{attrs:{"no-body":"","img-src":"/wiki/index.php?title=Special:Redirect/file/T--UiOslo_Norway--supervisors.jpg","img-alt":"Image","img-top":""},scopedSlots:e._u([{key:"header",fn:function(){return[t("h4",{staticClass:"mb-0"},[e._v("The Supervisors")]),t("b-card-sub-title",[t("br"),e._v("Prof. Dirk Linke "),t("br"),e._v("Dr. Athanasios Saragliadis "),t("br"),e._v("Kirsten Borse Haraldsen ")])]},proxy:!0}])},[t("b-card-body",[t("b-card-text",[e._v(" Prof. Dirk Linke runs a successful research group which focuses on the structure and function of the bacterial cell surface."),t("br"),e._v(" Dr. Athanasios Saragliadis is a postdoctoral research fellow in Prof. Linke's group, where he investigates the structure-function relationship in bacterial adhesins."),t("br"),e._v(" Kirsten Haraldsen is a research librarian in biosciences, with extensive expertise is sourcing collaborations and overseeing student projects. 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