Team:SDSZ China/Notebook
Notebook
These are what we did during the project. Click on the dates to see more information.
May-July
What we were doing in this period of time is to search for teammates from all kinds of school, from within Beijing to other areas, from China to America...
Another thing we did was to seek for a topic. This took quite a while, from selecting from a list of topics to investigating the feasibility of the topic.
8.9-8.11
8.9
1. Preparation
- Prepare LB medium, LB plates and experiment materials. (accidently add agar into liquid medium, will redo it tomorrow)
2. Inoculation
- Inoculate MG1655 bacteria into LB plate (without antibiotics).
8.10
1. LB medium Remake
- Remake two 1L LB medium (liquid) and LB plate (solid), use autoclave pans to sterilize the medium; prepare 10 LB solid plates without antibiotics and 20 more LB solid plates with antibiotics (both Amp+ & Kan+, 10 plates each); store all solid plates into 4℃ refregirator for future use.
2. Competent E.coli MG1655
- Make competent E.coli MG1655 cells using overnight cultured bacteria from yesterday (only to step 3, still need to culture it overnight and finalize the preparation tomorrow).
8.11
1. Done with Competent MG1655 cells
- Finish making competent E. Coli MG1655 cells, distribute them and store them into -80℃ freezer for future use.
8.12-8.14
8.12
1. PCR (MG1655 + PTarget)
System;
Colony PCR
25ul 2*Taq
2.5ul+2.5ul F+R
1ul Temple
19ul ddH2O
50ul Total
Primer annealing temperature 55℃
Extension 45s
FastPfu PCR
10ul Buffer
4ul dNTPs
2ul+2ul F+R
1ul Temple
1ul Enzyme
30ul ddH2O
50ul Total
Primer annealing temperature 57℃
Extension 75s
Colony PCR
25ul 2*Taq
2.5ul+2.5ul F+R
1ul Temple
19ul ddH2O
50ul Total
Primer annealing temperature 55℃
Extension 45s
FastPfu PCR
10ul Buffer
4ul dNTPs
2ul+2ul F+R
1ul Temple
1ul Enzyme
30ul ddH2O
50ul Total
Primer annealing temperature 57℃
Extension 75s
2. Gel Electrophoresis
1.2g Agarose
120ml 1*TAE Buffer
10ul SYBR safe DNA Gel Stain
120ml 1*TAE Buffer
10ul SYBR safe DNA Gel Stain
3. DNA Extraction
Concentration (ng/ul)
C1 9.35
C2 46.85
C3 16.15
C4 19.75
F1 9.45
F2 7.35
F3 12.75
F4 12.95
C1 9.35
C2 46.85
C3 16.15
C4 19.75
F1 9.45
F2 7.35
F3 12.75
F4 12.95
4. Gibson Assembly
5. Transformation & Inoculation
Transform Pcas into MG1655; Gibson-1 and Gibson-2 plasmids into Trans-5alpha.
- Transform Pcas plasmid into MG1655 competent cells (with K+ antibiotics), incubate at 30℃ for store it into 4℃ refregirator overnight; Transform Gibson plasmids into Trans-5alpha competent cells (with A+ antibiotics), incubate at 37℃ for overnight culture.
- Transform Pcas plasmid into MG1655 competent cells (with K+ antibiotics), incubate at 30℃ for store it into 4℃ refregirator overnight; Transform Gibson plasmids into Trans-5alpha competent cells (with A+ antibiotics), incubate at 37℃ for overnight culture.
8.13
Check bacteria growth, and store 2 plates of Pcas + MG1655 into 4℃ refrigerator.
1. Silicatein Transformation
- Transform Silicatein-pUC57 plasmid into LB solid plate with Amp+.
2. Silicatein Inoculation
3. Streaking plates
- For two Gibson + Trans5alpha 1 and 2 strains, make two streak plates (with A+ antibiotics) and incubate the plate into 37℃ incubator for overnight culture.
- For CsgA +Mfp5s with S010 S011 S021 strains, make three streak plates (with K+ antibiotics) and incubate them into 37℃ incubator for overnight culture.
- For CsgA +Mfp5s with S010 S011 S021 strains, make three streak plates (with K+ antibiotics) and incubate them into 37℃ incubator for overnight culture.
8.14
1. Colony PCR
2. Gel Electrophoresis
50ml system:
50ml 1*TAE Buffer
0.5g Agarose
5ul Stain
Results:
50ml 1*TAE Buffer
0.5g Agarose
5ul Stain
Results:
8.15-8.17
8.15
Got the sequencing results from yesterday: we selected sample 4, 7, 9, 10 for futhur analysis.
1. Inoculation
- Inoculate cultured bacteria (sample 4,7,9,10) from 4 Gibson-assembled pTarget-trans5alpha streak plates (see products from 8/13/2021) into 3mL of LB liquid medium (with Amp+ antibiotics), incubate it overnight on a shaker at 30°C.
- Inoculate cultured bacteria (single colony) from 2 pCas + MG1655 solid plates (see products from 8/12/2021) into 5mL of LB liquid medium (with K+ antibiotics), incubate them overnight on a shaker at 30°C.
- Inoculate cultured bacteria (single colony) from 2 pCas + MG1655 solid plates (see products from 8/12/2021) into 5mL of LB liquid medium (with K+ antibiotics), incubate them overnight on a shaker at 30°C.
8.16
1. pTarget Plasmid Extraction
- Making 4 PTarget plasmid extraction from yesterday's PTarget + Trans5alpha E. coli mixture (with Amp+ antibiotic). Store 4 PTarget plasmid at -20℃ freezer.
8.17
1. Making Pcas + MG1655 Competent Cells
- Transfer 0.1ml bacterial fluid (pCas) into 10ml LB (ADD K+ antibiotics!!!!)
- Cultured the strain at 30℃ for 1~2 hour, until OD600 grew to about 0.2
- Transfer 100ul 0.02% Arabinose into the strain
- Cultured the strain at 30℃ for 1.5 hour, until OD600 grew to about 0.2 - 0.25
- Immediately place it on the ice in an ice bath for 10 ~ 15 minutes to keep the bacteria in a cold environment.
- Transfer culture media into a precooled 50ml centrifuge tube, at 4℃ 4100rmp for 10min
- Add 6ml volume of cacl2-mgcl2 mixture into each tube, resuspend the cells and ice bath for 10min
- Store at -80℃
- Cultured the strain at 30℃ for 1~2 hour, until OD600 grew to about 0.2
- Transfer 100ul 0.02% Arabinose into the strain
- Cultured the strain at 30℃ for 1.5 hour, until OD600 grew to about 0.2 - 0.25
- Immediately place it on the ice in an ice bath for 10 ~ 15 minutes to keep the bacteria in a cold environment.
- Transfer culture media into a precooled 50ml centrifuge tube, at 4℃ 4100rmp for 10min
- Add 6ml volume of cacl2-mgcl2 mixture into each tube, resuspend the cells and ice bath for 10min
- Store at -80℃
2. pT Plasmids Transformation
- Transform pTarget plasmids extracted from yesterday into 4 different Pcas + MG1655 competent cells.
- Innoculate 4 tubes of Pcas+MG1655+pTarget bacteria mixture onto 4 plates with both Amp+ Kan+ antibiotics; incubate them at 30℃ overnight without a shaker.
- Innoculate 4 tubes of Pcas+MG1655+pTarget bacteria mixture onto 4 plates with both Amp+ Kan+ antibiotics; incubate them at 30℃ overnight without a shaker.
8.18-8.20
8.18
1. Streaking plates
- For 4 plates with 4 transformed pT plasmids, make 4 streak plates (with A+ K+ antibiotics) and incubate the plate into 30℃ incubator for overnight culture.
- Streaking method see below image:
- Streaking method see below image:
8.19
1. Colony PCR
- Colony PCR for pCas + pTarget + MG1655 engineered bacteria (using bacteria from streaked plates from yesterday)
- Preparation & System:
- Preparation & System:
- Operation:
1. Prepare the system, including the control group (MG1655 without any engineered plasmids or enzymes)
2. Use pippette or vortex mixer to blend the system. Then use centrifuge for 1.5minutes to make the mixture concentrate on the bottom of PCR tubes.
3. Use PCR apparatus to duplicate templete
4. Use electrophoresis apparatus to examine whether we knockout csgA successfully.
- Result:
1. The results did not quite meet expectations. The control groups works fairly well, whereas the experimental groups fall to form stripes, which indicates the strain is not what we cultivate. Luckily, we find the reason: previous templete shows that the earliest known problem appeared in the process of transforming MG1655 to csgA.
1. Prepare the system, including the control group (MG1655 without any engineered plasmids or enzymes)
2. Use pippette or vortex mixer to blend the system. Then use centrifuge for 1.5minutes to make the mixture concentrate on the bottom of PCR tubes.
3. Use PCR apparatus to duplicate templete
4. Use electrophoresis apparatus to examine whether we knockout csgA successfully.
- Result:
1. The results did not quite meet expectations. The control groups works fairly well, whereas the experimental groups fall to form stripes, which indicates the strain is not what we cultivate. Luckily, we find the reason: previous templete shows that the earliest known problem appeared in the process of transforming MG1655 to csgA.
2. Kill Switch Bacteria Plates
- Bacteria strains: BN063, BN065
- Method: streak the bacteria liquid mixture onto the 4 plates
- Incubate the 4 plates at 37℃ for overnight culture
- Method: streak the bacteria liquid mixture onto the 4 plates
- Incubate the 4 plates at 37℃ for overnight culture
3. Remake MG1655 LB Solid Plates
- Due to the unclear results from PCR of engineered bacteria, we had to redo the entire "gene knockout" process, unfortunately :(((
8.20
1. Silicatein Assay Solution Preparation
Metol-sulphite Solution
- Add 0.6 g of anhydrous sodium sulphite to 50 mL of Milli-Q water.
- Add 1 g of p-methylaminophenol (Metol solution).
- Once reagents are dissolved, filter through No. 1 Whatman paper and store in a glass bottom
- Recommended shelf life of approximately 1 month.
Oxalic Acid Solution
- Shake 5 g of oxalic acid dihydrate in 50 mL of Milli-Q water.
- Store in a glass bottle. Solution may be stored indefinately.
- Add 0.6 g of anhydrous sodium sulphite to 50 mL of Milli-Q water.
- Add 1 g of p-methylaminophenol (Metol solution).
- Once reagents are dissolved, filter through No. 1 Whatman paper and store in a glass bottom
- Recommended shelf life of approximately 1 month.
Oxalic Acid Solution
- Shake 5 g of oxalic acid dihydrate in 50 mL of Milli-Q water.
- Store in a glass bottle. Solution may be stored indefinately.
2. Remaking MG1655 Competent cells (step 1)
- Innoculate single colony from plate of MG1655 strain into 3mL of LB liquid medium (with NO antibiotics), put it into 37℃ on a shaker for overnight culture.
3. Kill Switch Innoculation
- Innoculate single colony from BN063, BN065 strains plates into 3 mL of LB liquid medium (with 30 μL K+ antibiotics)
- Incubate the liquid mixture at 37℃ on a shaker for overnight culture.
- Incubate the liquid mixture at 37℃ on a shaker for overnight culture.
4. Silicatein Innoculation
- Innoculate single colony from selicatein-puC57 strains plates into 3mL of LB liquid medium (with 30 μL A+ antibotics)
- Incubate the liquid mixture at 37℃ on a shaker for overnight culture.
- Incubate the liquid mixture at 37℃ on a shaker for overnight culture.
8.21-8.23
8.21
1. Kill Switch Testing
- Using 2 plates of LB solid medium (with K+ antibiotics), divide each plate into 4 sections, add 10uL of BN063 and BL065 cultured bacteria mixture into each section. (top 2 quarters without Ara, bottom 2 quarters with the addition of Ara Inducer droplet)
- Remark: Ara droplet addition: 30 uL; let Ara droplet completely dry before adding bacteria.
- Again, using 4 centrifugal tubes of LB liquid medium (1mL each), add 10uL of K+ antibiotics into each tube; add 10uL of BN063 and BL065 cultured bacteria mixture into each centrifugal tube. Then, choose 2 tubes and add 10uL of Ara Inducers into these 2 tubes.
- INCUBATE @ 37℃ OVERNIGHT CULTURE FOR ALL PLATES + TUBES.
- Why we use Ara inducer droplet?
- Remark: Ara droplet addition: 30 uL; let Ara droplet completely dry before adding bacteria.
- Again, using 4 centrifugal tubes of LB liquid medium (1mL each), add 10uL of K+ antibiotics into each tube; add 10uL of BN063 and BL065 cultured bacteria mixture into each centrifugal tube. Then, choose 2 tubes and add 10uL of Ara Inducers into these 2 tubes.
- INCUBATE @ 37℃ OVERNIGHT CULTURE FOR ALL PLATES + TUBES.
- Why we use Ara inducer droplet?
- Theory: When arabinose exists, AraBAD promotor(already in the bacteria) can drive the high efficiency expression of target genes which can translate into toxic protein to kill bacteria.
- Advantages:
1. Rigour
2. High inducibility
3. Low cost
- Advantages:
1. Rigour
2. High inducibility
3. Low cost
8.22
1. Kill Switch Testing
Result of BN063
8.24-8.26
8.24
1. Silicatein Assay Solution Preparation
Sulfuric Acid Solution
- Add 500 uL of Milli-Q water to 500 uL of concentrated sulfuric acid solution. (Final concentration of sulfuric acid in solution is 50%; final volume of the solution is 1mL)
- Store in bottle.
TMOS + HCl Solution Mixture
- Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) (1.47mL) in 1 mM HCl (3.1uL) to prepare prehydrolyzed silica particles.
- Calculation:
- 100mmol/L TMOS * 152 g/mol = 15.2g/L; (15.2 g/L) / (1.03 g/mL) = 14.7 mL/L ---> 14700 uL/L ---> 1470 uL/100mL ---> 1.47mL / 100 mL
- 1mmol/L HCl * 36.5 g/mol = 0.0365 g/L; (0.0365 g/L) / (1.18 g/mL) = 0.0309 mL/L ---> 31uL/L ---> 3.1uL / 100mL
Acidic solution of ammonium molybdate
- 20 g (NH4)6Mo7O24•4 H2O (0.33g) and 60 mL of concentrated Hcl [36%]
- WILL REDUCE THE ENTIRE SYSTEM INTO 1mL INSTEAD to minimize cost.
- Calculation:
- 20g/(2.5 g/mL) = 8mL
- 8mL : 60mL = 1 : 7.5 = 0.13mL : 1mL = 0.33g
- So, if we want to use only 1mL of concentrated HCl, we need to weight out 0.33g ammonium molybdate and mix them into solution for further use.
- Add 500 uL of Milli-Q water to 500 uL of concentrated sulfuric acid solution. (Final concentration of sulfuric acid in solution is 50%; final volume of the solution is 1mL)
- Store in bottle.
TMOS + HCl Solution Mixture
- Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) (1.47mL) in 1 mM HCl (3.1uL) to prepare prehydrolyzed silica particles.
- Calculation:
- 100mmol/L TMOS * 152 g/mol = 15.2g/L; (15.2 g/L) / (1.03 g/mL) = 14.7 mL/L ---> 14700 uL/L ---> 1470 uL/100mL ---> 1.47mL / 100 mL
- 1mmol/L HCl * 36.5 g/mol = 0.0365 g/L; (0.0365 g/L) / (1.18 g/mL) = 0.0309 mL/L ---> 31uL/L ---> 3.1uL / 100mL
Acidic solution of ammonium molybdate
- 20 g (NH4)6Mo7O24•4 H2O (0.33g) and 60 mL of concentrated Hcl [36%]
- WILL REDUCE THE ENTIRE SYSTEM INTO 1mL INSTEAD to minimize cost.
- Calculation:
- 20g/(2.5 g/mL) = 8mL
- 8mL : 60mL = 1 : 7.5 = 0.13mL : 1mL = 0.33g
- So, if we want to use only 1mL of concentrated HCl, we need to weight out 0.33g ammonium molybdate and mix them into solution for further use.
8.25
1. Research on TEM Sample Preparation
- Need to order "Carbon Membrane Supporting Film" and "Copper Mesh" for TEM testing
- Size: 200, 50 - Use a centrifuge to separate calcium hydroxyphosphate - Sample, disperse it in absolute ethanol - Ultrasonic dispersion - Drop on the Carbon Membrane Supporting Film(on the Copper Mesh) - Put them into an ice box
- Size: 200, 50 - Use a centrifuge to separate calcium hydroxyphosphate - Sample, disperse it in absolute ethanol - Ultrasonic dispersion - Drop on the Carbon Membrane Supporting Film(on the Copper Mesh) - Put them into an ice box
2. Silicatein-pUC57 Plasmid Extraction
8.26
1. Redo "Gene Knock-Out"
1. Making pCas+MG1655 Competent Cells------Protocol see above (2021/8/17).
2. Transform 4 pTarget plasmid into pCAS+MG1655 cells------Protocol see 2021/8/17.
2. Transform 4 pTarget plasmid into pCAS+MG1655 cells------Protocol see 2021/8/17.
2. Silicatein Plasmid Assembly
1. Plasmid Extraction of S010 Backbone
2. Fast Pfu PCR
10ul Buffer
4ul dNTPs
2ul+2ul F+R
1ul Template (5289 bp + 1459 bp = 6748 bp)
1ul Enzyme
30ul ddH2O
50ul Total
Primer annealing temperature: 56℃
Extension: 2 min 45 s
10ul Buffer
4ul dNTPs
2ul+2ul F+R
1ul Template (5289 bp + 1459 bp = 6748 bp)
1ul Enzyme
30ul ddH2O
50ul Total
Primer annealing temperature: 56℃
Extension: 2 min 45 s
3. Gel Electrophoresis
1. Add 0.3 grams of agarose, 30mL of TAE buffer and 3uL of SYBR safe DNA Gel Stain
2. Put the gel together with the plate into the electrophoresis tank and pour 1✖️TAE buffer onto the gel surface
3. Add 6x Loading Dye of 8 μL in each sample
4. 50 μL of S010, silicatein-pUC57 and DNA marker(Trans2K Plus II) were added respectively (total of 5 lanes-----S1, S2, P1, P2, Marker)
5. @ 150V, run the Gel Electrophoresis for 15min
Results:
1. Add 0.3 grams of agarose, 30mL of TAE buffer and 3uL of SYBR safe DNA Gel Stain
2. Put the gel together with the plate into the electrophoresis tank and pour 1✖️TAE buffer onto the gel surface
3. Add 6x Loading Dye of 8 μL in each sample
4. 50 μL of S010, silicatein-pUC57 and DNA marker(Trans2K Plus II) were added respectively (total of 5 lanes-----S1, S2, P1, P2, Marker)
5. @ 150V, run the Gel Electrophoresis for 15min
Results:
- All of the fragements have been successfully made.
- S1, S2 (base pair 1459 ✅)
- P1, P2 (base pair 5289 ✅)
- Moreover, we find out that Lane S1, S2 and P1 gave the best results.
- S1, S2 (base pair 1459 ✅)
- P1, P2 (base pair 5289 ✅)
- Moreover, we find out that Lane S1, S2 and P1 gave the best results.
4. DNA Extraction
Concentration ratio
- Silicatein-pUC57 : S010 = 3:1
- Silicatein-pUC57 : S010 = 3:1
8.27-8.29
8.27
1. Gibson Assembly
2. Transformation
- Transform Gibson-3 plasmid into BL21 E.coli strain (with K+ antibiotics), incubate at 37℃ for overnight culture.
8.29
1. Streaking plate (MG1655 Gene Knockout)
- Checking bacterial growth, found out only plamid pT#4 has successfully grown bacteria.
- For that plate with transformed pT#4 plasmids, make streak plate (with A+ K+ antibiotics) and incubate the plate into 30℃ incubator for overnight culture.
- Streaking method see below image:
- For that plate with transformed pT#4 plasmids, make streak plate (with A+ K+ antibiotics) and incubate the plate into 30℃ incubator for overnight culture.
- Streaking method see below image:
2. Streaking plate (Si)
- For Gibson-3 (Silicatein-pUC57 & S010) + BL21, make two streak plates (with K+ antibiotics) and incubate the plate into 37℃ incubator for overnight culture.
8.30-9.3
8.30
1. Colony PCR for MG1655
- Colony PCR for pCas + pTarget + MG1655 engineered bacteria (using bacteria from streaked plates from yesterday)
- Preparation & System
- Preparation & System
2. Colony PCR for Si
3. Gel Electrophoresis
50ml system
50ml 1*TAE Buffer
0.5g Agarose
5ul Stain
Results
Failed. Nothing ran out except the control group: MG1655 without any plasmids inserted, meaning that out experiment was NOT successful. Had to debug and redo it.
50ml 1*TAE Buffer
0.5g Agarose
5ul Stain
Results
Failed. Nothing ran out except the control group: MG1655 without any plasmids inserted, meaning that out experiment was NOT successful. Had to debug and redo it.
8.31-9.3
1. Redo Gene Knockout for MG1655
- Day 1: Using the MG1655 LB plate, innoculate single colony into LB liquid medium for overnight culture
- Day 2: Then make MG1655 competent cells; then transform pCas plasmid into it; overnight culture
- Day 3: Then making pCas+MG1655 competent cells; then transform pT(x4) into them, overnight culture.
- Day 4: After overnight culture, select single colony of pCas+pT+MG1655 cells and do Colony PCR for testing and sequencing (to see if we finally knocked csgA gene out OR not).
- Day 2: Then make MG1655 competent cells; then transform pCas plasmid into it; overnight culture
- Day 3: Then making pCas+MG1655 competent cells; then transform pT(x4) into them, overnight culture.
- Day 4: After overnight culture, select single colony of pCas+pT+MG1655 cells and do Colony PCR for testing and sequencing (to see if we finally knocked csgA gene out OR not).
2. Redo Gibson Assembly
- This time with the correct DNA concentration calculated by Nanodrop.
Concentration (ng/ul):
- S1: 31.45
- S2: 5.65 (disgard)
- P1: 34.25
- P2: 44.15
Concentration (ng/ul):
- S1: 31.45
- S2: 5.65 (disgard)
- P1: 34.25
- P2: 44.15
3. Transformation of Gibson-4 into BL21
4. Colony PCR + Gel Electrophoresis for Gibson-4 + BL21
- Select 8 single colony from plate of Gibson-4 + BL21, then run colony PCR to test out if any of the colony can success.
- DNA Marker: 100bp Plus II DNA Ladder
- Results for 8 Colony of G-4 + BL21:
- DNA Marker: 100bp Plus II DNA Ladder
- Results for 8 Colony of G-4 + BL21:
- Colony #4 successfully run out, meaning that this one matched with the fragment we desire. We will be using this colony to do PCR again tomorrow for both "backbone" and "fragment". And then run Gel Electrophoresis to test out if we successfully have them as the ones we designed.
- Results for backbone + fragment: (same marker as above)
- Results for backbone + fragment: (same marker as above)
- The results are positive(about 600bp)!!
- We sent both of them for sequencing, and the results for sequencing came out positive!! Now our Gibson Assembly for Silicatein + S010 strain are officially DONEEEEE!
- We sent both of them for sequencing, and the results for sequencing came out positive!! Now our Gibson Assembly for Silicatein + S010 strain are officially DONEEEEE!
9.4-9.6
1. Silicatein Activity Assay
- Prepare a solution of 100 mM tetramethyl orthosilicate (TMOS) in 1 mM HCl to prepare prehydrolyzed silica particles. Stir at room temperature for 15 minutes to completely hydrolyze TMOS to silica monomers.
- Prepare two reactions:
- Prepare two reactions:
- Incubate at NIST standard temperature and pressure (1 atm, 20 ºC) for 2 hours.
- Centrifuge sample at 15,000 RPM for 2 minutes to precipitate polymerized silica particles.
- Decant supernatant containing unaggregated silica particles.
- Wash 3 times with distilled H2O to remove free, hydrolyzed TMOS.
- Aliquot the unreacted hydrolyzed TMOS remaining in solution after silica aggregation.
- Treat with 2 M NaOH for 1 hour at 80 ºC to ensure complete hydrolysis of silica particles to monomer/dimer state.
- Remove aliquots of 500 μl and add to solution of 750 uL water and 75 uL of acidic solution of ammonium molybdate (20 g (NH4)6Mo7O24•4 H2O and 60 mL of concentrated Hcl [36%]).
- Incubate at NIST standard temperature and pressure for 20 minutes. Solution should have a yellowish hue after incubation.
- Add 400 uL of reducing agent solution (see above). Solution should turn a bluish color.
- Record absorbance at 810 nm and compare to standard curve.
To make sure we have valuable comparison, in our own experiment apart from the previous protocol, we designed:
- two negative control groups----NC1, NC2;
- two experiment groups----USE ultrasonic homogenizer; NO ultrasonic homogenizer
Data:
- Qualitative Pictures:
- Centrifuge sample at 15,000 RPM for 2 minutes to precipitate polymerized silica particles.
- Decant supernatant containing unaggregated silica particles.
- Wash 3 times with distilled H2O to remove free, hydrolyzed TMOS.
- Aliquot the unreacted hydrolyzed TMOS remaining in solution after silica aggregation.
- Treat with 2 M NaOH for 1 hour at 80 ºC to ensure complete hydrolysis of silica particles to monomer/dimer state.
- Remove aliquots of 500 μl and add to solution of 750 uL water and 75 uL of acidic solution of ammonium molybdate (20 g (NH4)6Mo7O24•4 H2O and 60 mL of concentrated Hcl [36%]).
- Incubate at NIST standard temperature and pressure for 20 minutes. Solution should have a yellowish hue after incubation.
- Add 400 uL of reducing agent solution (see above). Solution should turn a bluish color.
- Record absorbance at 810 nm and compare to standard curve.
To make sure we have valuable comparison, in our own experiment apart from the previous protocol, we designed:
- two negative control groups----NC1, NC2;
- two experiment groups----USE ultrasonic homogenizer; NO ultrasonic homogenizer
Data:
- Qualitative Pictures:
NC1 (for NP) = Negative Control Group #1 (only TMOS + HCl)
NC2 (for P) = Negative Control Group #2 (TMOS + HCl + PBS buffer)
NP = NO ultrasound homogenization (TMOS + HCl + NaOH + silicatein extracted from 5mL of original bacterial mixture)
P = Under ultrasound homogenization (TMOS + HCl + PBS buffer + NaOH + silicatein extracted from 5mL of original bacterial mixture)
NC2 (for P) = Negative Control Group #2 (TMOS + HCl + PBS buffer)
NP = NO ultrasound homogenization (TMOS + HCl + NaOH + silicatein extracted from 5mL of original bacterial mixture)
P = Under ultrasound homogenization (TMOS + HCl + PBS buffer + NaOH + silicatein extracted from 5mL of original bacterial mixture)
Level of colorness: NP < P < NC1 < NC2
Level of transparency: NC2 < P< NP < NC1
- Qualitative OD values:
Level of transparency: NC2 < P< NP < NC1
- Qualitative OD values:
The results show that there are sufficient amount of silicatein produced, since:
- Both NC Control are darker (NC1 remain as yellow which indicates that it needs MORE NaOH to turn blue, it contains more acids)
- Both silicatein mixture (with / without homogenization) have higher OD810nm, meaning that there are more bacteria inside the test tube (the solution is thicker / not as clear as NCs).
- Both NC Control are darker (NC1 remain as yellow which indicates that it needs MORE NaOH to turn blue, it contains more acids)
- Both silicatein mixture (with / without homogenization) have higher OD810nm, meaning that there are more bacteria inside the test tube (the solution is thicker / not as clear as NCs).
2. Silicatein Miniralization Products Extraction (used for TEM/SEM testing)
- We use the 80 degree Celcius dryer to try to heat-dry the silicatein protein-bacteria mixture (5mL), in hope that there will be products produced after 1~2 days of physical drying. If the results are prominent, we can move on and send the products for TEM/SEM testing.
- Results (left: control; right: silicatein after drying)
- Results (left: control; right: silicatein after drying)
3. Incubation of Gene Knockout products
- At 30 degree Celcius, overnight culture
- Failed. There was little to no bacteria grown on the plate after 2 days of culture. We reached out to a previous team who had done experiments on Gene Knockout and MG1655 bacteria strain, and asked about their results, in hoping we could find a solution. They told us that their results for knocking out MG1655 was not obvious (shown no promoninet results after and before the knockout). Thus, we decided to change our experiments setup and skip the gene knockout part, and to use the bacteria directly for our next step testing.
- Failed. There was little to no bacteria grown on the plate after 2 days of culture. We reached out to a previous team who had done experiments on Gene Knockout and MG1655 bacteria strain, and asked about their results, in hoping we could find a solution. They told us that their results for knocking out MG1655 was not obvious (shown no promoninet results after and before the knockout). Thus, we decided to change our experiments setup and skip the gene knockout part, and to use the bacteria directly for our next step testing.
9.9-9.14
9.9
1. Innoculation again.
- Innoculate Silicatein, S010, S011, S012 bacteria strain from plates to LB liquid medium
- Overnight culture with 37 degree Celcius on a shaker
- They are prepared to be used for TEM/SEM testing!
- Overnight culture with 37 degree Celcius on a shaker
- They are prepared to be used for TEM/SEM testing!
2. Prepared some more LB liquid medium.
9.10
1. Add inducer IPTG + innoculated bacteria mixture
- Added 100uL of IPTG and 200uL of 4 types of bacteria mixture(Si, S010, S011, S021) from yesterday respectively, into 4 different glass bottles containing 200mL of LB liquid medium(with K+ antibiotics), put them into 37 degree Celsius incubator on a shaker for overnight culture.
- Concentration of IPTG inducer is 5mmol/L, thus we need to use ratio 1/2000 to add inducer into LB medium to induce growth; and use 1/1000 ratio to add bacterial mixture into LB medium to make sure normal growth as well.
- Concentration of IPTG inducer is 5mmol/L, thus we need to use ratio 1/2000 to add inducer into LB medium to induce growth; and use 1/1000 ratio to add bacterial mixture into LB medium to make sure normal growth as well.
9.11-9.14
1. Preparing for TEM + SEM testing
- Prepare 5 pieces of “Quantifoil Carbon Film”.
- TEM testing:
- Draw 1mL of Si bacteria mixture from the original LB medium glass bottle into microcentrafigal tube. Add 10uL of bacteria mixture onto the piece of carbon film (mark as #1).
- Dilute the original Si bacteria mixture with ratio 1:10; add another 10uL of bacteria mixture onto another piece of carbon film, repeat it twice (mark as #2, #5).
- Repeat the step above twice, mark as #3, #4.
- Put all 5 pieces of film into the 80 degree Celsius dryer until they are completely dry, then send them for TEM testing.
- Pictures:
- Draw 1mL of Si bacteria mixture from the original LB medium glass bottle into microcentrafigal tube. Add 10uL of bacteria mixture onto the piece of carbon film (mark as #1).
- Dilute the original Si bacteria mixture with ratio 1:10; add another 10uL of bacteria mixture onto another piece of carbon film, repeat it twice (mark as #2, #5).
- Repeat the step above twice, mark as #3, #4.
- Put all 5 pieces of film into the 80 degree Celsius dryer until they are completely dry, then send them for TEM testing.
- Pictures:
- Results:
- SDS-PAGE verification:
- Draw 50mL of bacteria mixture from 4 different bottles from yesterday.
- Centrifuge 4 bacteria mixture (10000rpm, 15min), decant the supernant; for all of them, add 10mL of PBS buffer and use ultrasound to homogenize the mixture (10min each).
- Aliquot the supernants and filter the liquids.
- Run SDS-PAGE to separate and test protein.
- Results:
- Draw 50mL of bacteria mixture from 4 different bottles from yesterday.
- Centrifuge 4 bacteria mixture (10000rpm, 15min), decant the supernant; for all of them, add 10mL of PBS buffer and use ultrasound to homogenize the mixture (10min each).
- Aliquot the supernants and filter the liquids.
- Run SDS-PAGE to separate and test protein.
- Results:
SDS-PAGE Protein Gel Electrophoresis:
1. Prepare SDS-PAGE running buffer(1X): 10mL.
2. Prepare gels and assemble the electrophoresis device.
3. Heat samples for 5 minutes at 94 degrees Celsius.
4. Cover the chamber & firmly connect both the anode and the cathode. Set the voltage on the electrophoresis power supply to a constant voltage of 120V.
5. Run the gel electrophoresis cell for 30-60 minutes.
6. After electorphoresis, remove the gel by floating it off the plate into water.
7. Stain the SDS-PAGE with Coomassie blue dyes, then destain it.
8. Observe the electrophoresis results that show up in the E-gel imager.
1. Prepare SDS-PAGE running buffer(1X): 10mL.
2. Prepare gels and assemble the electrophoresis device.
3. Heat samples for 5 minutes at 94 degrees Celsius.
4. Cover the chamber & firmly connect both the anode and the cathode. Set the voltage on the electrophoresis power supply to a constant voltage of 120V.
5. Run the gel electrophoresis cell for 30-60 minutes.
6. After electorphoresis, remove the gel by floating it off the plate into water.
7. Stain the SDS-PAGE with Coomassie blue dyes, then destain it.
8. Observe the electrophoresis results that show up in the E-gel imager.
