Team:UZurich/Notebook
Notebook
Notes
06/28
First Day in the Lab!
Inoculation of BL21 strain and BL21-Omp deletion strain to 5ml LB buffer (3 each).
37 degree Celsius culturing overnight.
06/30
Redo the transformation for all six samples (pSB1C3, pSB3C3, pSB3K5, pSB4A5, pSB4C5, pSB4K5) following the protocol
Inoculation of colonies from a few strains' plates and preparation for the DNA extraction and genotyping.
(O)MVs harvesting
OD600 measuring for six samples (diluted with water by 10 fold) (see Table 1).
Preparation of the solutions for centrifuge: If the weight of the liquid is not equal, add water (samples can vary maximally by 1g)
Centrifuge the sample: 8000 RMP at 4 degrees for 20 min --> gets rid of debris and some of DNA
Filtering with falcon pump the samples' solution and storing them in bottles. The samples treated with MMC showed a more sticky property and changing filtering paper is necessary. (Another option is using a vacuum filtering system). Make sure to wash the filter apparatus after every sample
Centrifuging the solution, removing the supernatant, and getting (O)MVs from the precipitate. The condition is 150,000x g, 4 ℃ for 1h. Make sure the samples are balanced(using LB buffer). And this step was done three times to obtain all (O)MVs.
After centrifuging, resuspend the precipitate by mili-Q water and centrifuge again in the same condition.
Discard the supernatant and resuspend the precipitate with different amounts of water according to the (O)MVs we obtained from step 4 (see table 2).
Storing the concentrated (O)MV sample in freezer.
07/01
DNA extraction following the protocol: see Miniprep Protocol.
Inoculation of colonies from all six plates(from yesterday's transformation) and prepare
for genotyping.
DNA density measurement: using NanoDrop.
The principle is to measure the light absorption from purine base and pyrimidine base which is around 260nm.
We sometimes can also see the peak from ethanol(~220nm) and from protein(~280nm)
First open the program and choose Nucleic Acid.
Adding water(1.5 µl) and do the initialisation
After initialisation using water to do the calibration(set the baseline)
Then using tissue to clean the detector.
Put 1.5 µl sample on the detector and start the measurement.
Record the concentration and A260/A280 ratio and clean the detector
Following the steps above and finish all the measurements.
Result is shown in table 1.
07/02
Plans:
(O)MVs quantification
Miniprep the inoculated samples from 01.07(6 samples)
Restriction enzyme treatment and run the gel to separate the linear backbone
If we have microsynth's order we can do further PCR reaction.
(O)MVs quantification
Protein quantification: using BCA protein quantification kit
Preparation of standard calibration with BSA: 2000µg/ml, 1000µg/ml, 500, 250, 125, 62.5, 31.25, 15.625, etc.(details can be found in excel file later) --> have a known absorbance curve (give us the linear curve in image 1, data points represent the absorbance of our own samples)
Mix the reagent A and reagent B(A:B = 50:1).
Preparation of sample: 1µl sample + 9 µl water + 10 µl 4% SDS + 180 µl reagent mix (reagent mix is added last!). (Depending on the concentration of our sample we can use different ratio of sample and water but in total 10µl)(for calibration we add 10 ul sample without water)
Incubation of the sample for 15-30min in 37℃ (watch the color change)
Do the quantification at absorption of 562nm.
Using the calibration data to do the linear regression.
Fit the sample data to this model and calculate the protein concentration.
Result is shown in table 2 and image 1.
Lipid level test: using FM™ 1-43
Dilute the FM 1-43 in 1:1000. (we mix 0.75 µl FM with 750 µl of water to obtain 1:1000)
Mix 1 µl sample with 100 µl of diluted FM 1-43. (The blank is made by only 100 µl of diluted FM 1-43)
And Quantify directly with exctiation in 485nm and emition in 590nm.
Result is shown in table 1.
Seedling growth inhibition with isolated vesicles
Question: What response do we get from the seedlings when treated with hight concentration vesicle treatment? Which treatment results in an ERF response in the seedlings?
Using 2 x 48 well plates
We left out the BL21 treatment for now
Using two strains of plants (efr-1, col-1)
Use MS as medium
Normalise vesicle samples by protein
End concentration of vesicles per well: 5 μg / 1 ml
Per well we fill in 500 μl
Volume of treatment solution: 5 μg/value from table 2 (μg/ml) --> gives the amount of ml of treatment per well (transform into μl by multiplying with 1000)
Dilute the vesicle samples (we had very small quantities of vesicles) 1:10 = vesicles:water --> 50 μl of vesicles and 450 μl of water (total 500 μl per well)
Handling seedling:
Desinfect workspace and hands
Use MS as base solution
Mix a tube with MS and amount above of treatment solution (don't forget to pipette out the same amount of MS out of the tube as is added from the treatment to prevent spilling)
Pipette 500 μl of the mix into the wells
Add seadling into every well
Seal the lid with breathable tape
Store in seedling room
Plasmid quantification from new samples:
Use 1.5 μl of water to apply onto the machine (name?)
Click on blank, then measure --> make sure output is about 0
Use 1.5 μl of the sample and note 260/280 ratio (protein to DNA ratio) and purity of the sample
Results
07/05
Plan:
Most likely we will spend all time on molecular cloning.
Purify the pSB backbone(Gel purification)
Making our pGGA' and pGGE' plasmid(Using primer: iGEM1_pGGA1_R(F) and iGEM1_pGGE1_R(F) for backbone and iGEM5_T7-lacO_F(R), iGEM7_T7Ter_F(R) as target)
Making dummy entry vectors
Starting Greengate reaction to anneal the entry vectors
During greengate we can do another reaction to insert clyA to the pSB backbone and if possible, start transformation.
Work
Digesting the backbone plasmids for Cly-A + Epitopes to get the linear backbone and cut out the useless fluorescence protein fragment.
Resuspend the dry DNA: ClyA, epitopes
Gel purification and DNA extraction for ClyA-Epitope backbones: pSB1C3, pSB3C5 and pSB4C5
Setting up a gel purification: pSB(1C3, 3C5 and 4C5) backbones (preparing the tray)
Unpacking the received synthesized DNA (Cly-A + Epitopes)
Following the protocol to prepare the dried DNA
We want to do a control gel purification with the undigested backbone (Cly A + Epitopes) aswell (so getting these aswell as the digested ones)
Preparing PCR tubes with all the backbones(for adding Cly A&Epitopes) for gel purification (20 mi/L 2x high copy number backbone (digested), 1x 5 mi/L high copy number backbone (undigested), 2x 20 mi/L medium copy number backbone (digested), 1x 5 mi/L medium copy number (undigested), 2x 20 mi/L low copy number backbone (digested), 1x 5 mi/L low copy number backbone undigested)
Setting up the gel electrophoresis in the Et-Br room
Adding loading dye (sample volume/ 5)
Adding DNA ladder 1000 bp
Loading the gel
Performing the gel extraction: using the knife to cut the band from the gel under the UV light and purification of the Cly-A + Epitopes gel according to the protocol on the E.Z.N.A. gel extraction kit (Nanodrop result see table 1)
PCR reaction for making pGGA' (pGGE') backbones: 32µl dH2O, 10µl Greenbuffer, 5µl dNTPs, 1µl iGEM1_pGGA(E)1_R, 1µl iGEM1_pGGA(E)1_F, 10ng of template and 1µl Polymerase.
Setting up another gel purification for the T7 Promoter/Terminator and RBS/Terminator backbone
Performing the gel extraction and purification of the T7 and RBS backbone gel according to the same protocol
Prepare the working solution of the primers and T7, T7T oligos in 1:10 dilution.
Making a mixture of T7(R and F, 10 mili liter each and 80 mili liter of water to get another 1:10 dilution), T7T (Same as in T7), and two dummies(same as in T7)
Putting the fragments for insertion into the PCR for annealing (Temperature slowly decreasing from 97.5 to 10 in steps of 2.5)
For pGGA & pGGE green gate we add (T7 Promoter or A' (Dummy) for pGGA and T7 Terminator or E' (Dummy) for pGGE), 8 miL of Fr 1 miL T4 Ligase, 1 miL BsaI, 1 miL T4 Ligase buffer, then fill up to either 10, 15 or 20 miL with water (whichever needs the least addition of water)
For the Cly-A Golden Gate we add (4 miL pSB1C3 or 3 miL pSB3C5 or 1 miL pSB4C5), 8 miL Cly-A fragments (annealed (ds)), 1 miL T4 Ligase, 1miL BpiI, 1.5 miL T4 Ligase buffer, then we fill up to either 10 miL, 15 miL or 20 miL with water (whichever needs the least addition of water)
For time-saving reasons additionally to producing the entry vectors in the PCR (takes 3h) we parallel produce the pSB1C3 plasmid with T7 Promoter, RBS, Venus (NLS), Venus, Venus, Terminator, T7 Terminator directly with the dsDNA and let it run overnight (less efficient).
07/06
Work
Before lunch:
Making a gel (useless though...)
Take the PCR tubes and do the transformation (Four PCR tubes are used for the transformation):
The first one is pSB1C3 as backbone and T7, T7 terminator and pGGA-pGGE as entry "vectors"(we use T7 and T7 terminator in dsDNA form)
The second one is pSB1C3 as backbone and two dummies at the begin and the end and pGGA-pGGE as entry "vectors" (we use two dummies in dsDNA form)
The third one is pSB4C5 as backbone and T7, T7 terminator and pGGA-pGGE as entry "vectors"(we use T7 and T7 terminator in dsDNA form)
The last one is is pSB4C5 as backbone and two dummies at the begin and the end and pGGApGGE as entry "vectors" (we use two dummies in dsDNA form)
The transformation follows the protocol and this time we use 7.5µl of DNA
After lunch:
Seed sterilisation: 3 eppendorf tubes each for efr-1 and col seeds and put them into the sterilisation chamber with 50 ml 5% bleach and hcl (this starts a reaction that forms chlorine gas) leave them for 4 hours
After transfer the seeds (dispersed pay attention that there are no clumps) onto MS plates, close them with breathable tape and put into cold room in the dark box for stratification (we can put them into the grow room thursday or monday and need to use them 4 days after that).
Also discard the eppendorf tubes into biowaste bc can be seeds stuck to the wall
Also we ordered soil plates and need to collect them on thursday (for the ROS assay)
Label the white label with iGem and put a yellow (bc arabidopsis-transgenic) in each of the pots stating the genotype (we need to mix the genotypes)
- finished the transformation and put onto 3 plates, 2 for 1C3 and one with T7 and blank for the 4C5
- redoing plates for clyA for pSB4C5 and pSB3C5 because they didnt grow (spreading 150 microliters this time)
07/07
Plan
Redo series from 05.07 and 06.07 (received no results)
Do digestions
Do PCR and gel extraction/purification with pGGA/E and 1C3, 4C5, 3C5
Annealing ssDNA to dsDNA
Gel extraction of pGGA'/E'
Tube weights:
A': 0.4 g
E': 0.6 g
Follow OMEGA protocol in Gel Extraction Kit
Quantify the extracted DNA with nano drop:
Gel extraction of 4C5, 3C5, 1C3
Weight of tubes:
4C5: 0.1 g
3C5: 0.1 g
1C3: 0.1 g
Quantification
Annealing
Use R and F primers from primerbox: Tubes 5-14
Dilute primers by 10 with water
Ratio small fragment:backbone = 3:1
Calculate concentration of annealed solution: We need to insert 1 ul dsDNA into pGGA'/E'
Starting the golden gate reaction
Starting liquid cultures with the 1C3 vectors at different antibiotic concentrations: 0.5, 1, and 5 miL
07/08
Work
Doing the transformation of Cly-A into the 1C3, 4C5 & 3C5 backbones.
Doing the transformation of the T7 promoter and terminator and 2 dummies into the pGGA and pGGE backbones.
Doing a mini-prep for 1C3 cultures at different concentrations of antibiotics
We planted the seedlings into the soil and put them into ARALAB 18 (need to remove the lid tomorrow (09.07) will be ready in 3 weeks (29.07))
Prepared a 10 mL culture with K antibiotic of the strain: JW3132 nlp1 for making competent cells out of it tomorrow.
Measured concentrations of all the unaltered backbone stocks from iGEM (Only the C backbones) and picked the highest concentration for sequencing
We took the 1C3 5 miL liquid culture as well as the 4C5 and 3C5 strains and prepared it for sequencing
We filled out the in-house sequencing form and sent it to Lena Stransfeld (Order is 1C3 13, 3C5 13, 4C5 13, 1C3 14, 3C5 14, 4C5 14)
07/10
Work
Made competent cell with the nlp-1 knock-out strain. Did 1/10 of the recipe.
Tested the competent cells via transformation with the 4C5 backbone. Results tomorrow. Added 1 microliter of DNA).
Ligated PGGA backbone to A' and T1 Promoter and the PGGE to the E' fragment and the T7 Ter
Transformed the result from the ligation with the normal competent cells.
07/12
Work
Greengate reaction to build up a vector: backbone: pGGZ; entry vectors: pGGA-pGGF. (1 µl of each vector, 1 µl BsaI, 1 µl buffer and 1 µl water)
Do the transformation using this resulting vector to competent Ecoli cells.
Colony PCR of colonies from plate: pGGA' and pGGE'. Three colonies from each plate were chosen.
Run a gel of colony PCR result. Three bands were get from pGGA' colonies. After comparing with the theoretical result, we decided to do a sequencing to these colonies tomorrow and therefore we inoculate these three colonies.
Growth inhibition tests are done and we scaled the seedlings.
We decided to isolate OMVs from our strains(Lpp deleted strains) so we start with a small scale inoculation.
New primers and strategies are designed for fusion-detection vectors.
07/13
Work
Extract plasmids from yesterday's pGGA' inoculation and do a squencing.
New primers are designed and purchased for synthesis.
Briefl analyzed the data from the seedlings growth inhibition test.
Run the growth curve of the strain: BL21, Omp, NlpI, rseA, degP, TolA and TolB using yesterday's culturing.
Re-inoculate these seven strains for tomorrows OMV isolation.
07/14
Work
Analysis of data from growth curve experiment.
Plasmid isolation of pGG: pGGZ as backbone and pGGA-F as entry vector.
pGG plasmid digestion: using 4 µl plasmid, 1 µl KpnI, 1 µl XbaI, 3 µl 10 x buffer(with BSA) and 21 µl water. After 2h digestion run the gel. Result will be uploaded. (Plan to use primers to do the PCR and get the middle pGGA-F part for justification)
Preparation of glycerol stock bacteria strains: BL21, Omp, NlpI, rseA, degP, TolA and TolB
Test the OD 600 of the strains we want to use for tomorrow's OMV isolation and inoculated them to a 200ml culture erlenmeyer. The OD 600 for each strains will be uploaded later.
Plasmid isolation: pGGZ as backbone, pGGA-F as small fragment
Nanodrop readout:
Digest the plasmids into a linear strand (with Kpn1 and Xba1) and run through electrophoresis
Verdict: The golden gate method is suitable to build the designer plasmids (verify with Philipp)
Create new OMV's with new strains
OD600 of the following strains in small culture
Dilute to 0.01 OD600 in 200 ml LB: 200x0.01/OD600 = ml
Attention: We diluted to 0.02 OD in 200 LB for our 200 ml culture
Transfer the diluted amount of small culture bacteria into a 200 ml culture of LB and let grow overnight
07/15
Plans
Isolate OMV from the new stains: omp8, degP, RseA, TolB, TolA, Nlp1
Following workflow from the 30.06.21 Lab Journal
Work
Optical density of the 200 ml bacteria culture [OD600]:
Omp: 3.77
Nlp1: 5.69
RseA: 5.08
deg P: 4.37
Tol A: 6.17
Tol B: 4.46
Isolation
Samples were centrifuges three times and resuspended, then frozen
Tol A and resA didn't show a pellet
07/16
Plans
Wash and centrifuge and resuspend the OMV samples from yesterday
Do lipid and protein quantification for the new OMV samples
Do lipid quantification for the old OMV samples (last time we only did protein quantification)
Work
OMV sample preperation
Resuspend the pellets in following volumes:
Nlp1: 200ul
Omp8: 200ul
TolB: 200ul
deg P: 50ul
rseA: 230ul
TolA: 160ul
Protein quantification
Prepare reagent mix: 3000ul of reagent A and 60ul of reagent B (total volume: 3060ul)
Add 1 ul of sample and 9ul of water (in total 10 ul), add 10ul of 4% SDS
Add 180ul of reagent mix
Tip: If bubbles emerge, poke them with a tip
Incubate for 15 to 30 min
(more details: see Lab Journal 02.07.21)
Lipid quantification
Use column 5 of the welled plate
Dilute the FM 1-43 in 1:1000. (we mix 0.75 µl FM with 750 µl of water to obtain 1:1000)
Mix 1 µl sample with 100 µl of diluted FM 1-43. (The blank is made by only 100 µl of diluted FM 1-43)(for sample C and F we add 5ul instead of 1)
And quantify directly with exctiation in 485nm and emition in 590nm.
Make lipid normalisation for the old OMV samples
Use Omp sample for normalisation
Dilute the following samples by 10 (some are already diluted, some need to be diluted again):
PAO1 MMC, BL21 MMC, omp, omp MMC
Volumes of 10 fold diluted samples added to the MS medium:
Pao1 MMC: 199.3ul
BL21 MMC: 425.7ul
omp: 199ul
omp MMC: 458.9ul
Make dilution series for the new samples
For a selected sample (we selected omp) we begin with 50ul and dilute in steps of
07/19
Plans
Looking for viral vectors on the internet
Culturing different bacterial strains for comparison of MV production and fusion:
Agrobacterium tumefaciens grows at 28°C optimally
Pseudomonas putida IsoF (leo eberl) grows at 30°C optimally
Pseudomonas syringae grows at 28°C optimally
Pseudomonas fluorescens CHAO (leo eberl) grows at 30°C optimally
Bacillus subtilis 168wt (PxylA-xhlAB-xlyA) (leo eberl) grows at 37°C optimally
Preparing cultures for competent cells BL21 omp8 and TolB and BL21 wt
Work
Prepared the culture of BL21 omp8 for competent cells, made two 5 ml liquid cultures (one from old BL21 omp8 plate and one from the new one) put it into the shaker at 14.30
Prepared 6 seedlings (3x efr & 3x col0) and put them into the stratification box
07/20
Plans
Create competent cells from over night culture
Molecular cloning if primers arrive
Receiving shop card from Lena
Work
Preparing compentent cells of BL21 wt, BL21 omp8 & TolB (following the protocol of the lab)
We got the shop card
Getting the bacterial strains from Leo Eberls Lab stocks on our prepared salt-free agar plates (P.Putida, P. Fluorescence and B. Subtilis) (Will arrive next morning)
Preparing agar plates with Rifampin (Rif) & Gentamicin (Gent) for the strain A. tumefaciens and Rifampin (Rif) & Kanamycin (Kan) for the strain P. Syringae (We will get both strains from Kyle)
Bought stuff at the lab
We need to order the following:
Pierce™ BCA Protein Assay Kit
Durapore® Membranfilter, 0,45 µm
Received the primers from Microsynth
Started with the molecular cloning
Following the PCR protocol:
Prepared a gel and performed gel electrophoresis of the PCR product.
iGem 18 & iGem 19 together with 1C3 or 4C5 did not work
Gel extraction of the ones that did work
Redoing the PCR for the faulty ones (iGem18 with 1C3 and iGem 19 with 4C5 and iGem 22 & 23 with 3C5)
To one of the new PCR products (6) we added the restriction enzyme DPN1 to cut the methylated backbone (which is not a PCR product)
07/21
Plan
Gel preparation for 3 & 4 which did not work (now with adjusted Tm)
Transformation of 1C3 & 4C5 + Cly-A
For PCR Nr. 6 which is iGem 22 and iGem 23 + 3C5 of the gel extracted DNA of the first
PCR from yesterday and with the new one which is digested to clean it from residues
Getting the bacterial strains (P.Putida, P. fluorescence and B. subtilis from Leo Eberls lab)
Creating a bacterial stock for these strains
Work
Gel preparation and gel electrophoresis (Let it run for 30min)
Transformation of 1C3 & 4C5 + Cly-A
Getting the bacterial strains from Leo Eberls lab
Gel extraction of PCR products 3 & 4 following the protocol
The concentrations are 41..5 ng/miL and unknown purity for PCR product 4 and 25.8 ng/miL and purity 2.02 for PCR product 3
07/22
Work
We got colonies from the transformations we did yesterday exept for 4C5 Cly A with the normal comp cells and 6 gel extracted one
Colony PCR on the Cly A constructs so 1C3 cly A Tol B , 4C5 Cly A Tol-B and 1C3 Cly A with normal comp cells
Vol 25 mil 16 h2o 5 green buffer 2.5 dNTP 0.5 VF2, 0.5 VR o,5 X7 Polymerase
Transformation of the Green gate constructs songyang did yesterday
1C3 PT7 35S omega enhancer NLS 3x Venus hsp18 T7Ter
1C3 PT7 ClyA Flag 22
4C5 PT7 Cly a elf18
4C5 PT7 35S omega enhancer NLS 3x Venus hsp18 ter T7ter
Made new Cm plates
redid Rif+Kan plates bc p. syringae didnt grow so tryed again is now in the 28 incubater
Made Glycerol stocks of B.Subtilis, p. fluirescence CHAO and p. putida
Made a gel withe the colony PCR product
Inoculated 3 replicates of the 6 with dnp I and the without Dnp 1 (overnight 5ml culture in the shaker)
Inoculated agrobacterum strain 5 ml overnight culture to make glycerol stock tomorrow
07/23
Plans
Prepare A. tumefaciens glycerol stock
Order soil
Put 1 plate of each seed in to the grow room to put in soil on Tuesday
Work
Did miniprep on our overnight cultures
Results yield purity
4C5 Cly A -1 169.1 1.79
4C5 Cly A -2 148.2 1.78
4C5 Cly A -3 153.2 1.66
1C3 ClyA -3 109.6 1.72
1C3 ClyA -2 165.2 1.63
1C3 ClyA -1 65.0 1.72
1C3 35S 1 15.4 1.47
1C3 35S 2 74.4 1.76
1C3 35S 3 211.4 1.82
3C5 Bsa1 Mut -1 367.2 1.84
3C5 Bsa1 Mut -2 241.1 1.81
3C5 Bsa1 Mut -3 96.6 1.71
3C5 Bsa1 MutDpn1 -1 243.5 181
3C5 Bsa1 Mut Dpn1-2 219.3 1.81
3C5 Bsa1 Mut Dpn1 -3 186.6 1.82
Testing these:
For clyA constructs via PCR
React. vol. 25 mil (16 h2o, 5 green buffer, 2,5 dNTP, o.5 VF 2, 0.5 VR, 0.5 X7 Polymerase
For the other two via digestion
For 35S: react vol 10 mil (2 mil plasmid (over 200 ng), 1 mil Spe1, 1 mil Xba1, 1 mil cutsmart Buffer, H2O)
For the BsaI constructs (IGem 6):
React vol. 10 mil. (over 200 ng plasmid, 1 mil Bsa1, 1 mil T4 Ligase buffer, H2O)
After run gel to see if they are correct
Put 1 plate of Col-0 and efr-1 seeds into grow room
Made agrobact. glycerol stock
Doing transformation with Tol-B comp cells with golden gate constructs from yesterday
(1C3 PT7 35S omega enhancer NLS 3x Venus hsp18 T7Ter
1C3 PT7 ClyA Flag 22
4C5 PT7 Cly a elf18
4C5 PT7 35S omega enhancer NLS 3x Venus hsp18 ter T7ter)
Ordered two soil trays and booked the aralab 18 for them
Running the gel of the digestion
Running the gel from the PCR (if bands look right will prepare glycerol stocks of the overnight liquid cultures)
Inoculated the colonies from plates:
(1C3 PT7 35S omega enhancer NLS 3x Venus hsp18 T7Ter
1C3 PT7 ClyA Flag 22)
07/26
Plans
Evaluate seedling growth inhibition
Maybe start some OMV isolation
Work
Did miniprep on the 5 ml cultures of 1C3 T7 35S 4-8 and the 3C5 gel extraction Bsa1 mutant.
Results: yield purity (260/280)
gel+Dpn1 3C5 Bsa1 mut 163.2 1.88
1C3 T7 35S 4 126.0 1.76
1C3 T7 35S -5 94.1 1.70
1C3 T7 35S -6 134.7 1.82
1C3 T7 35S -7 48.4 1.75 29.0 1.68 32.3 1.84
1C3 T7 35S -8 47.1 1.68 100.4 1.53
For the 7 and 8 re did the elution and left them on the 60 degrees for couple minutes to evaporate the ethanol before centrifuging again.
Started 5 ml liquid culture of p. syringae with gent and rif to make glycerol stock later
Ran the gels for the miniprep products and other BsaI mut from last week for BsaI bands seem to be correct but we need sequencing to be sure. For the others gel looks wrong so we redid the green gate reaction.
Also we did seedling weighing for the seedling growth inhibition.
07/27
Work
Sequencing:
Inoculation of strains for OMV and MV harvesting: Omp8, P.fluorescence, P.putida, P.syringae, agarobacteria, B.subtilis.
Mainly discussion.
07/28
Plans
Meeting with C.Zipfel
Transfering the seedlings (Efr-1 & Col-0) into soil
Preparing bigger cultures for OMV isolation of the different strains (A. tumefaciens, P.syringae, P. putida, P. fluorescence CHAO, B. subtilis 618, E. coli BL21 omp8)
Redo the construction of the linearized backbone with T7 promoter and T7 terminator and redo the Green Gate reaction to create the fusion testing construct
Work
Meeting with C.Zipfel
Transfering the seedlings (Efr-1, Col-0) to soil
Received more seeds from Kyle (Col-0, Efr-1, Fls-2 & Efr-1 & Cerk-1 (tripple-mutant called fec))
PCR of the linearized backbone with T7 promoter and T7 terminator
Measuring OD600 of our bacterial strain liquid cultures and calculating the amount that we need to add to the 2 x 200ml flasks (one for OMVs and one for CMVs):
Formula: (Volume of target flask * desired OD600)/Measured OD600
BL21 omp 8 new plate: 6.28 => transfer 0.64 ml into 200ml flask to reach OD600 of 0.02
BL21 omp 8 original plate: 0.98 => transfer 10.2ml into 200 ml flask to reach OD600 of 0.05
BL21 omp 8 glycerol stock: 3.21 => transfer 1.25ml into 200ml flask to reach OD600 of 0.02
P. flouresence: 1.51 => transfer 2.65ml into 200ml flask to reach OD600 of 0.02 (we only have 5ml so we take 2 times 2.5ml)
P. syringae: 0.33 => transfer 30.3ml into 200ml flask to reach OD600 of 0.05 (we only have 5ml so we take 2 times 2.5ml)
P. putida: 1.18 => transfer 3.39ml into 200ml flask to reach OD600 of 0.02 (we only have 5ml so we take 2 times 2.5ml)
A. tumefaciens: 4.78 => transfer 0.84ml into 200ml flask to reach OD600 of 0.02
B. subtilis: 2.85 => transfer 1.4ml into 200ml flask to reach OD600 of 0.02
Let 200 ml culture sit for 3 hours
After 3 hours add MMC to all the samples(except for p.syringae since the concentration is too low)
Doing the gel extraction of the linearized plasmid: Concentration later
Phosphorylating the ends of the linearized plasmid to make it circular(Using T4PNK, detailed amount later)
Run another Greengate reaction using linear 1C3 backbone and other 5 entry vectors.
Transformation of pGGC to amplify the plasmid
07/30
Plan
Miniprep overnight cultures
Make Green Gate and transformation with overnight culture
Isolate OMV's
Work
Morning:
Zetrifuge OMV and CMV samples
Miniprep overnightculture via protocol provided by promega
Afternoon:
Protein quantification
Prepare reagent mix: 3000ul of reagent A and 60ul of reagent B (total volume: 3060ul)
Add 1 ul of sample and 9ul of water (in tottal 10 ul), add 10ul of 4% SDS
Add 180ul of reagent mix
Tip: If bubbles emerge, poke them with a tip
Incubate for 15 to 30 min
(more details: see Lab Journal 02.07.21)
Lipid quantification
Use column 5 of the welled plate
Dilute the FM 1-43 in 1:1000. (we mix 0.75 µl FM with 750 µl of water to obtain 1:1000)
Mix 1 µl sample with 100 µl of diluted FM 1-43. (The blank is made by only 100 µl of diluted FM 1-43)(for sample C and F we add 5ul instead of 1)
And quantify directly with exctiation in 485nm and emition in 590nm.
Miniprep
Nanodrop results:
08/02
Plans
Miniprep of 1C3 T7 mVenus construct in normal DH10 cells
Digestion for analysis of the construct using Xba1 & Spe1 restriction enzymes
Gel electrophoresis for testing if we see two bands
Reading the article
Work
Did the miniprep of the 1C3 T7 mVenus
Did the digestion for analysis
1: 35.7, 1.80/25.4, 1.69
2: 100.3, 1.73
3: 111.1, 1.79
4: 96.3,1.82
5: 76.6, 1.79
6: 90.8, 1.81
7: 124.2, 1.76
PGGD: 176.4, 1.80
Doing the Gel electrophoresis
Gel had no EtBr in it, so redoing the digestion
Preparing chemicals for a ROS assay
Getting tobacco plants for ROS assay
Doing an HR test on N.benth
1 = P.fluorescence CMV high
2 = P.fluorescence OMV high
3 = A.tumefaciens CMV high
4 = A.tumefaciens OMV high
5 = P.fluorescence CMV 1/10 diluted
6 = P.fluorescence OMV 1/10 diluted
7 = A.tumefaciens CMV 1/10 diluted
8 = A.tumefaciens OMV 1/10 diluted
Results:
Following the CZL protocol for ROS assay
Doing a golden gate to ligate the 1C3 | 4C5 backbone and the Cly-A
08/03
Plans
Finishing the ROS assay
Work
Reading the paper
Preparing the ROS solution:
- 2ml MilliQ H2O per row
- 2miL HRP
- 2miL luminol | LO/12
- decide on concentration of OMV/CMV add 50 or 100 miL (same volume per MV)
- add everything to small falcon tubes
Doing the ROS with N. benthamiana and P.fluoresence OMV | CMV, A. tumefaciens OMV | CMV, E. coli BL21 omp8 OMV | CMV, Positive control flg-22, negative control H2O
- To reach a concentration of 10 mig/ml add:
- P.flourescence OMV 180 mil
- P.fluorescence CMV 18.3 mil
- A.tumefaciens OMV 371 mil
- A.tumefaciens CMV 27 mil
- E.coli BL21 omp8 OMV 52 mil
- E.coli BL21 omp8 CMV 27 mil
Sadly the ROS did not work (flg-22 response was too low) probably because N. benthamiana was rubbish
Moffat repeated the ROS in lettuce with our P.fluorescence CMVs and it showed a very strong response
Preparing new seedlings A. thaliana col-0 | efr-1 | efr-1 & fls-2 & sec-1 tripple mutant | bak-1/ 5
Getting leaf discs of Moffats lettuce for another ROS assay for tomorrow
Doing Miniprep of 1C3 bpi 1 -2 (1 ml DNA)| 1C3 Bpi 1-1 (3ml DNA) | pGGB (1 ml DNA):
- Bpi1-1: 76.6 ng/ul, 1.81
- Bpi1-2: 22.4 ng/ul, 1.96
- pGGB: 78.9 ng/ul, 1.90
Doing the golden gate for the different backbones from above with Cly-A
Preparing 1C3 T7 mVenus 2-5 and 1C3 T7 PNK 1 & 3 for sequencing
08/04
Plans
Do another ROS assay with P.fluorescence CMV, A.tumefaciens CMV | OMV, E.coli BL21 omp8 CMV|OMV, P.putida OMV
Meeting with Beat Keller to discuss HIGS/RNAi application to our project
Work
Did another ROS assay with P.fluorescence CMV, A.tumefaciens CMV | OMV, E.coli BL21 omp8 CMV|OMV, P.putida OMV
Meeting with Beat Keller
Results
Doing the miniprep from yesterday again (1C3 bpi 1 -2 (1 ml DNA)| 1C3 Bpi 1-1 (3ml DNA) | pGGB)
Nanodrop results:
- pGGB mVenus 268.6 ng/μl 1.66
- pGGD mVenus 175.7 ng/μl 1.85
- 4C5 Cly-A J23119 280.5 ng/μl 1.85
Colony PCR
Prepare 5ml overnight cultures of P.fluorescence, P.putida , P.syringae and E.coli omp8 for OMV harvest
08/05
Plans
Start OMV/CMV isolation
Sequencing results of the GreenGate (Fusion reporter construct) are bad
Doing the GreenGate again, but screening many more colonies (80-100 instead of 8) Moffat advised us
Preparing P. syringae glycerol stocks
Using E. coli BL21 omp8 competent cells for the Cly-A construct (maybe that works better)
Our T7 Promoter - Backbone - T7 Terminator construct didn't work as well (but we also didn't screen many)
- Alternative would be to use HBLC or Page purified primers
Meeting with Caua
Work
Doing a new round of GreenGate for the fusion reporter casette construct and screening more colonies
Meeting with Caua:
- Order sequences with IDT:
o Order 1: T7 Promoter - 35S - Ω-TMV - 3x mVenus - HSP-18 - T7 Terminater
o Order 2: ESAR system with GFP with TEV recognition site (flanked by restriction sites) and replaceable TEVp (flanked by restriction sites)
o Order 3: Cly-A construct with inducible promoter or weaker constitutive promoter
o Order 4: ESAR system combination of Tol-B repressible and TEVp inducible
Use Microsynth for DNA sequencing
- We need labels (Caua has many of them at Irchel)
- There is a box for sending it in (also at irchel)
- If we don't use PCR products, we need special tubes (need to order them in the future but Caua has some for now)
Started a PCR for the T7 Promoter-Backbone-T7 Terminater for sequencing with Microsynth
Prepared a glycerol stock of P. syringae
Decided on which OMVs/CMVs to isolate tomorrow (2x P. syringae CMV and OMV, 2x P. fluorescence OMV, 1x E. coli BL21 omp8 OMV and CMV)
Bringing PCR product to Caua for sending to sequencing
08/09
Plans
Get omp-A plasmid
Do plasmid miniprep
SZ explain Cly-A construct
Check induced resistance
- Prepare P. Syringae
- MV treatment
IDT Order (first check with Caua)
Contact Microsynth because of repeated delays
3C5 mut seq
Work
Got omp-A plasmids
Did Miniprep
Contacted Microsynth because of delays: Delays due to quality control (they do it again if quality is bad and ship everything together)
08/18
Work
Did an other infusion experiment with p. fluorescence CMVs and BL21 CMV to see what concentrations wont kill the cells
p. fluorescence start 7200 prot conc and dilute 10 times 3 times so 720 72 and 7,2
For BL21 start at 2800 to 280, 28 and 2,8
The highest concentration wasn't filtered
We use 4 N. benth plants and one leaf each with all the four treatments and one with 4 MQ infusions as negative control.
Also put seedlings into soil from bak 1-5, efr-1, fec and Col-O genotypes
08/19
Work
Prepared the newly arrived primers and their dilutions.
Did miniprep on the new arrrived parts.
Nanodrop 19.08.21
08/23
Plans
Weighing the seedlings for SGI
Doing another round of MV/OMV isolation
Preparing sequencing
Doing miniprep
Work
Weighed the seedlings for SGI
Did another round of OMV/MV isolation
Prepared the sequencing with Microsynth
08/30
Work
Miniprep different constructs:
L1 tolB 1: 29.4/1.66
Flg elf 2: 65.9/1.72
L1 dCBD 2: 62.3/1.79
L1 TEV 2: 68.8/1.56
L1 TEV1: 17.7/1.58
L1 tolB 3: 36.4/1.89
Flg elf 3: 74.3/1.77
L1 dCB3: 67.7/1.87
L1 TEV 3: 23.9/1.69
L1 dCBD 1: 54.4/1.83
Flg elf 1: 135.0/1.71
L1 tolB 2: 52.4/1.72
Isolate OMV's from tolB constructs:
A1: tol B cly A elf18 1
B2: tol B cly A elf18 2
C3: tol B cly A flg22 1
D4: tol B cly A flg22 2
E5: tol B clyA only
F6: tol B clyA only no inducer
Get agrobacteria with plasmids from Prof. Voinnet:
- 35S:GF-FG in binary vector pBin61 obtained in Agrobacterium on Petri dish
- 35S: PVX210DIelta25 (GFP-tagged potato virus X minus movement and coat proteins) in binary vector pBin61 in obtained in Agrobacterium on Petri dish
- 35S: P19 in binary vector pBin61 obtained in Agrobacterium on Petri dish
Not forget that growth conditions differ for Agrobacterium (28°C, supplemented with rifampicin) and E.coli (37°C)
Innoculate HT115 (DE3) and L4440
08/31
Work
Make competent cells from HT115 (see protocol CZL booklet)
Miniprep L4440
Plants are ready for ROS --> ROS done next week
Make SDS gel for western blot
Innoculate 3 agrobacteria strains with different vectors each (from Voinnet):
- 35S:GF-FG in binary vector pBin61
- 35S: PVX210Dlelta25 (GFP-tagged potato virus X minus movement and coat proteins) in binary vector pBin61
- 35S: P19 n binary vector pBin61
Sequence the following (three groups removed due to low concentration):
(L1 tolB 1) -> removed
Flg elf 2: 65.9/1.72
L1 dCBD 2: 62.3/1.79
L1 TEV 2: 68.8/1.56
(L1 TEV 1) -> removed
L1 tolB 3: 36.4/1.89
Flg elf 3: 74.3/1.77
L1 dCB3: 67.7/1.87
(L1 tev 3) -> removed
L1 dCBD 1: 54.4/1.83
Flg elf 1: 135.0/1.71
L1 tolB 2: 52.4/1.72
Sequencing (Micro Synth):
1: L1 dCBD 1
2: L1 dCBD 2
3: L1 dCBD 3
4: L1 TEV 2
5: L1 TolB 2
6: L1 TolB 3
7: flg elf 1
8: flg elf 2
9: flg elf 3
Miniprep L4440
Results Nanodrop:
L4440 1: 9
09/01
Plan
Do western blot
Miniprep plasmid out of agrobacteria and transform them into DH10B:
35S:GF-FG in binary vector pBin61
35S: PVX210Dlelta25 (GFP-tagged potato virus X minus movement and coat proteins) in binary vector pBin61
35S: P19 n binary vector pBin61
Prepare leave discs from col-0 and erf-1 for ROS
Inoculate sequenced colonies from yesterday in 5ml culture:
Colonies: flg22-elf18-1, flg22-elf18-3, dCBD-1, dCBD-2, TEV-2, tolB-2, TolB-3
Nanodrop Results from Miniprep
35S:GF-FG in binary vector pBin61: 35.3 ng/ul and 280/260 1.74
35S: P19 n binary vector pBin61: 66.3 ng/ul and 280/260 1.70
35S: PVX210Dlelta25: 64.6 ng/ul and 280/260 1.65
Transformation of the plasmids we minipreped into DH10B
Follow Protocol
Using: 2ul of plasmid
Western blot
Samples: TolB ClyA with inducer, TolB ClyA without inducer, TolB ClyA-flg22 with inducer, TolB ClyA-elf18 with inducer, omp8 Cly A with inducer, omp8 ClyA without inducer, omp8 ClyA-flg22 with inducer, omp8 ClyA-elf18 with inducer
Mix samples with loading dye
ROS
Make LO/12 stock solution
2mg of Lo/12 g in 6.4 ml MQ
09/02
Work
ROS test using vesicles: tolB-ClyA-No inducer, tolB-ClyA, tolB-flg-1, tolB-flg-2, tolB-elf-1, tolB-elf-2
LMU goldengate: Para-ClyA-elf-flg, Para-ClyA-dCBD, Para-tolB, Para-TEV, Positive control and transformation
Transforamtion of BB05-BB09
Send sequencing
09/06
Work
Miniprepped see below
Sent sequencing of the things we miniprepped beforehand
Prepared the samples for the fusion test at the eth following their protocol
09/07
Work
Prepared to redo the Ros assay with n. beth so we took leaf discs from 4 plants for one 96 well plate
Molecular cloning
First prepared gBlocks then set up the PCR reaction, see image below
Filtered and rediluted deltaTolB ClyA only induced CMVs for the ROS assay today we got a plate of Col-O to run from a friend who doesnt need it today, so we can run it.
09/08
Work
Autoclaving bottles for OMV isolation
ROS test
Transformation of uTEV, eTEV and tolB with 3x TEV recognition site
09/09
Work
OMV isolation of Cly-A uninduced, Cly-A induced, Cly-A elf-18 -1, Cly-A elf-18 -2, Cly-A, flg-22 -1, Cly-A flg-22 -2
PCR of GFP quencher for TevP testing
Gelextraction of the PCR product
09/10
Work
Had meeting about the presentation video in the morning
Miniprepped and sent for sequencing following samples:
Went to the ETH to look at our infiltrated leaves with the confocal microscope (infiltrated with PVX and P19)
We didn't see anything for the CMVs but for the agrobacterium (positive control) and for the E.coli!!
We will discuss the results with professor Voinnet on tuesday at 2 pm but this may indicate that e. coli can transfer DNA into plant cells somehow or this is just an anomalie related to it being a viral vector.
Took leaf discs for ROS assay two plates of col-0 and one plate efr-1 to test the engineered vesicles again.
09/13
Work
Miniprep of Para Cly-A elf-18 flg-22, dCBD
PCR
Gelelectrophoresis (Constructs incorrect except 1,2 and 8)
Restriction-Digestion test (All constructs incorrect)
09/14
Work
Transformed uTEV, eTEV, tolB, tolB TEV, TEV into DH10 competent cells and inoculated them over night in the 37°C incubator
SGI with Lore, fec & col-0
Went to ETHZ to meet with Prof. Voinnet but he wasn't there.
Looked at the plants at ETHZ but it was too soon to see anything meaningful
09/15
Work
SGI of plants treated with OMV's containing ClyA only, ClyA-flg22, ClyA-elf18
Colony PCR of uTEV, eTEV, tolB, tolB TEV, TEV
Subsequent gelelectrophoresis of the PCR product
Ladder, positive control, 5x TEV, 5x uTEV, 3x eTEV, Ladder
Ladder, positive control, 2x eTEV, 5x tolB, 5x tolB TEV, Ladder
Inoculation of the working strains (TEV, uTEV, eTEV, tolB, tolB TEV) in CARB LB
Inoculation of omp8 Cly-A only, omp8 Cly-A elf-18, omp8 flg-22 in CARB LB
09/16
Work
Video meeting in the morning
Plan experiments with Prof. Voinnet after lunch
Miniprep: tolB TEV, tolB TEV 4, tolB 4, tolB 2, tolB 1, eTEV 5, eTEV 4, uTEV 4, uTEV 3, TEV 7, TEV 6
Make 200ml culture from omp8 ClyA, omp8 flg22, omp8 elf18
omp8 flg22: add 1.56 ml
omp8 elf18: add 1.08 ml
omp8 ClyA: add 0.99 ml
09/20
Work
PCR of GFP-Quencher construct
Miniprep for workshop (failed)
09/27
Work
Miniprep of workshop plasmids
Transformation of omp-A flg-22 elf-18 dCBD constructs