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Team:Cambridge/Bacillus/Lab Work
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Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm. | Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm. | ||
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Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them! | Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them! | ||
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- Add 9μL of SDW, 5μL of MM, 1μL of VF, 1μL VR and 4μL of each biobrick (program iGEM new) | - Add 9μL of SDW, 5μL of MM, 1μL of VF, 1μL VR and 4μL of each biobrick (program iGEM new) | ||
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Latest revision as of 03:56, 30 October 2008
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July 22ndWe have 4 tubes from last year. These strains are frozen in glycerol.
Checking antibiotic resistancePurpose : Grow each of them on a plate to test their antibiotic resistance Protocol
July 23rd
1/ Purpose Check antibiotic resistance of our different strains
- Grow 15uL of B. 1A1 (frozen tube) in 5mL of LB - Incubate at 37°C 3/ Purpose : Grow plasmids in TOP10, transformation 4 plasmids :
1 control : PUC19 - Add 20uL of TOP10 competent cells and 0.5uL of Plasmid in an eppendorf - 30min on ice - Heat shock : 60s at 42°C - 2min on ice - Add 89.5uL of SOC - 60min at 37°C - Put the mix on plate with ampicillin resistance - Incubate at 37°C
July 24th
Antibiotic test for Bacillus resistanceWe want to find the lowest concentration of antibiotic which kills Bacillus.
Dilution of Cm. [ 1:1000 means 1 part stock to 1000 part Sterile Distilled Water ]
Results [Plates were prepared before lunch, at 5pm there were visible growth] -Amp Plates: Huge rings of no growth around 100, 75, 50, 25, and 10. -Cm PLates: Tiny rings of no growth around 5, 10, 15 and 25. Small ring of no growth around 35 but not well defined. (No clear zones)
Salts for making Bacillus Competent10x Medium A Base
-Aliquot into 5 different bottles. [180mL each] 10x Bacillus Salts
-Aliguot into 5 different bottles. [200mL each] Medium B
-Total volume 200mL NOTE: To complete Medium B, Take 0.2mL of this solution July 25thResults from Yesterday
Results for Amp Even with a concentration of 10μg/mL, Amp kills Bacillus. Since we want to know which is the lowest concentration of Amp which kills B.S, we are going to test with some lower concentrations. Results for Cm Even with a concentration of 35μg/mL, ther is not a clear area around the antibiotic disk. So we have to test some higher concentration of Cm. Wet Work
Dilution of Amp
- Melt Soft Agar - 3mL SA + 10μL Cells 1A1 from LB prepared on 23/7/08 - Pour SA+Cells over blank hard agar plates - Put 5 blank disks on each agar plate - Add 40μL of antibiotic on each disk - Incubate at 37°C Results Nothing! 1A1 cells were kept in the freedge! B.S. can not be kept in the fridge, low temperatures kill them! July 28th
For Cm, 35μg/mL should be enough to kill B.S. For Amp, nothing can be concluded! Wet Work
We had 2 samples of ppL82 in DH5α in LB solution (~4mL), one from the frozen glycerol tube and one from a colony picked on a plate. Normally plasmid and cells should not be kept in freezer. So, we want to extract this plasmid, check its size and then keep it in the fridge. We do that for the 2 different samples.
- Test the concentration of DNA in each tube
- Plate a single colony (from 23/07/08) on a Cm plate - Incubate at 37°C
- Incubate at 37°C
-Pick e single colony of MC1061 (from 22/07/08) and put it in 10mL of LB (to make glycerol stocks) -Incubate at 37°C
- Add 100μL of LB+1A (mix of friday) and 10mL of LB - Incubate at 37°C
- Prepare Medium A Add 81mL of SDW, 10mL of 10X Medium A base and 9mL of 10X Bacillus salts
 At, t0 = 70min, log growth seems to stop.
- Storage : we want to store 7 tubes 6 tubes in the freezer (with 60μL of glycerol) 1 tube on the bench - Transformation
July 29th
 The ladder seems to be wrong. So it was really difficult to ckeck the size of our plasmid ppL82. To make, that, we assume that the size of our biobricks was ok, and we estimate the size of our plasmid. The plasmid ppL82 seems to have the right size, but we will have to check again to be able to use a ladder.
We observed B.S. with a microscope. Problem, all B.S. seems to be dead! Possible reasons of this problem : - Problem with the vector (it could be a wrong vector, we are not really sure) - Quantity of DNA, we added 0.5μg. The protocol was 1μg, but normally it should be ok. - B.S. may not be competent (we have to test motility with microscope) - Cells could be dead : replate them - Try to add tryptophan in the medium
Wet Work
- Preparation of medium A with tryptophan : 81mL of SDW, 9mL of 10X Bacillus salts, 10mL of 10X Medium A base and 0.1mL of Tryptophan (11mg/mL)
- Digest : with PstI and SalI from Biolabs, and Buffer 3 (add 15μL of DNA) - Gel (17μL of sample and 3μL of dye)
Results from the gel
 The sizes we expected were about 1100kb and 2100kb. The sizes should be ok!
July 30th
Medium A
For this medium, the curb stop to increase logarithmically at 100min.
For this medium, the curb stop to increase logarithmically at 90min.
- Prepare 7 tubes for medium A and 7 tubes for medium A + tryptophan : add 0.45mL of prewarmed medium B and 0.05mL of culture and incubate 90min at 37°C by shaking. - Transform B.S. by adding DNA and incubate 30min at 37°C We have one transformation with ppL82 (add 7.5μL of DNA), one with PNZ8901 (add 10μL of DNA) and one negative control without DNA. - Plate 500μL on Cm plate (35μg/mL)
- Plamsid miniprep (for I746001, I746101 and PNZ8901 from big flasks - Measure DNA concentration in our samples to decide the volume of DNA we have to add in our preparation
 Everthing is really too big! There is a problem, either dimerization, either contamination, either a problem in our work. So we are going to run a new gel.
- Miniprep plasmid from growth bottles (I746001, I746101 and PNZ8901); from plates (I746001, I746101 and PNZ8901). - Do single digest for PNZ8901 (one with PstI, one with SalI) - Run a gel with : PNZ8901 from friday, PNZ8901 digest with PstI, PNZ8901 digest with SalI, 3 samples from growth bottles, 3 samples from plates (to check the size of the uncut vectors)
 The ladders are really bad! But the size of our biobricks and plasmid are too big! So we can not trust these big cultures. They is a problem. With tests from the first week, we know that biobricks are right, so we are going to grow new cultures from these first cultures, to make stocks. Concerning the vectors, they are from last year, so we are not sure of what they are. Since we ordered new well defined vectors (we should receive them on friday), we will use them in the next steps to be sure of our work.
August 1st
- Preparation of Agar plates We have bottles of 200mL.
We prepare 10mL of LB in which we add a single colony.
August 2ndYesterday's Results
Lab WorkFridged all plates except ECE 172 ==> Benched. All tubes palleted and placed in freezer although ECE 176, 172, 153 doesn't seem to have any pallets All red plates does not seem to have any extra growth despite the air conditioning had been turned off when I entered the lab this afternoon.
August 4thWet Work
- for each vector, plasmid miniprep from frozen pellets (step 7 only once, with 60μL of Elution Buffer for ECE112, ECE147, ECE149, ECE 150 and only 30μL for the others) - Single digest (15μL of SDW, 2μL of Buffer, 2μL of DNA, 1μL of enzyme, then incubate 10min at 37°C and 30 min for the one using HindIII with EcoRI buffer) - Double digest (mix 7.5μL of each single digest, and then incubate 10min (or 30min for EcoRI + HindIII and EcoRI buffer) at 37°C, then heat shock 5min at 80°C
 - ECE112, ECE165, ECE166, ECE171 : right bands, ok - ECE172 : no band, not enough DNA - ECE147, ECE150, ECE162 : only one band, maybe not enough DNA - ECE149 : wrong size! - ECE151 : not sure, check again
- Streak new plates with different strains of Bacillus : 1A1, IA751 and IA771
August 5thChecking vectors- Plasmid miniprep for ECE166 (for stock), ECE172 and ECE153 - Digest of ECE153, ECE172 and ECE162 (with plasmid miniprep from yesterday) - Run on a gel single digest for ECE147 (from yesterday with more DNA), ECE149 (from yesterday with more DNA), ECE150 (from yesterday with more DNA) and ECE 153, ECE162, ECE172
 Very low bands : not enough DNA!!! Transformation of Bacillus
- Prepare medium A with tryptophan, and medium B - add 5mL of medium A in 3 different tubes, inoculate each tube with colonies (1A1, IA751 ans IA771) - Check OD every 20min - Incubate 90min - Add 0.45mL of medium B and 0.05mL of culture in Ependorf tubes - Incubate 90min - Transform : add 1μg of DNA (some with ECE112, some with ECE166) (so you need to nanodrop samples before!) - Incubate 30min - Pipette 200μL of solution, spread it on aech plate, wait 10min, and do it again - Incubate 24hours - A few glycerol tubes to stock cells : add 2/7 glycerol to cell tubes
- Spin glycerol stocks, pipette out glycerol - Add 0.5mL of medium B, incubate for 1 hour - Add 10μL of ECE112 (640ng) - Incubate 2hours - Plate 200μL, and 10min later, still 200μL
New stocks- Do glycerol stock of I746001 and I746101 (no sterile glycerol) - Put IA751, IA771 in 10mL LB - Put ECE 176 in 10mL LB + antibiotic - Reinoculate the tube of LB from yesterday with ECE166 plate - ECE176 replated onto Amp100 + Cm5
August 6thResult from transformationPLATES HAD BEEN BINNED!! OOPS Check Vectors- Plasmid miniprep for ECE176 (from 05/08 LB stock and from 04/08 LB stock with a second inoculation) We want to check uncut plasmid, single and double digest for ECE147, ECE149, ECE150, ECE153, ECE162, ECE172, ECE176. In order to have enough DNA (to see the bands), we will add 1μg of DNA to make single digest, and wr will incubate during 1h (instead of only 10min).
We made singe digest for the samples from 05/08 only. - Double digest (1 hour of incubation in water bath at 37°C) - Gel Gel1
 Gel2
Our supercoiled ladder was very bad, so it was impossible to conclude for uncut vectors. - ECE147, ECE 150, ECE153, ECE 176 single digest : ok - ECE149 : problem, 2 cutting sites for PstI (only one in the sequence) - ECE162 : only one band whereas SalI should have 2 cutting sites - ECE172 : wrong sizes!!! - Double digest for ECE176 : ok! - Double digest for ECE 150, 149 and 147 : only one band!!! There may be a problem when we do the double digest by adding two single digest. We will try again with a direct double digest. August 7thTransformation of B.S. IA771New transformation with the same protocol than 2 days ago. We will try to add less liquid on plates (to avoid growing colonies in liquid)
Check VectorsWe want to make a final check for ECE147, 149, 150, 153 and 162. - Double digest + gel
 - ECE 147, ECE150, ECE153 :ok! - ECE 149 : 3 bands! not the right vector - ECE162 : ony one band! It has been uncut... maybe the good vector (according to precedent gel), but not sure Test ECE112 transformation- for the three different strain (1A1, IA771, IA751), make 12 spots - take one colony with a loop, and put it on a Cm5 plate and on a Cm5 + Spc100 plate (do that for the 12 spots) - incubate New stock- Put ECE171 in 10mL of LB - Incubate August 8thResult from yesterday transformation
Result for test of ECE112 transformationOn Cm5 plates, we have some colonies for each strains, on Cm5 + Spc 100 plates, no colonies on each plates. This result seems good, however, we forgot to make a control. Since, we had some colonies on control plates, it is possible that our bacillus are resistant to Cm5, even if they are not transformed. We addded some control colonies. Control with erythromycin- Prepare erythromycin, stock 5mg/mL : 0.05g in 10mL of SDW - Prepare plates : add 20μL of Ery (5mg/mL) into 200mL of agar (final concentration : 0.5μg/mL) - Do several spots on each plate
Amylase production screening
- Starch : 1g of starch in 10mL of SDW - Add 2mL of Starch solution into 200mL of agar, mix -Inoculate on different plates : 1A1 +ECE112 (plate from 05/08), IA751 + ECE112 (plate from 05/08) and IA771 + ECE153 New stocks- Take 1mL from LB stock oh ECE171 (from 07/08) and put it into 99mL of LB August 11thErytromycin Experiment- New preparation of Ery : 0.05g into 10mL of ETHANOL - We plated again our plates (same than 08/08/08) Amylase screening experiment- New method : add 2g of starch powder into 200mL of Agar, shake, pipette to plate (and avoid bubbles) - Same plates than 08/08/08 Test our stock of ECE171-Plasmid miniprep 9from the samples of 10mL and the one of 100mL) -Nanodrop both DNA
- Double digest (EcoRI and PstI) with 10μL of DNA and 1h of incubation - Samples have been frozen, they should be run onto a gel Control of Spc resistance of Bacillus- Spc50 plate with transformed IA771 (ECE153, which is Spc resistant) and with IA771 9which should not be Spc resistant) August 12thResults from yesterday
- IA751 (control) : most of colonies did not grow, which is good, but 2 or 3 grew... - IA7771 + ECE166 : all colonies grew : ok 9non integration vector) - IA771 + ECE153 : growth of all colonies... problem!!! if IA771 is transformed with this integration vector, it should lose its EryR - IA771 + ECE112 : growth of all colonies... problem!!! if IA771 is transformed with this integration vector, it should lose its EryR
- IA771 : no growth on Spc50 plate, so no natural resistance - transformed cells grew
- Add iodine solution for 1min - Problem : no aparent blue, no diffusion of iodine into LA agar + a lot of contamination New plates and LB stocks- In order to try to make plasmid miniprep at the end of the day, grow IA771 transformed with ECE166 in 10mL LB + Cm5 7hours is not enough to reach exponential phase!!! We will try again tomorrow by incubating longer - Grow IA771 and IA751 ( first plate from sterile disks of strain) imto 10mL of LB - Grow ECE166, 171, 153 in E.coli with approriate antibiotics 9to transform tomorrow) - Grow IA771 transformed with ECE153 into LB+Spc50 (to check fluorescence with xylose induction)
New test for amylaseWe tried 2 different methods. - Dilute 1g of starch into 100mL of agar and try to dilute it and boil it to sterilize. The problem is that it is very difficult to dilute... - The second method seem to be better. We diluted 1g of starch into 100mL of Soft Agar (it dilutes very well). Then plate blank agar plate (LA) and then add a thin layer of SA ith 1% of starch. Poke the plates. - We plated IAI+ECE112, IA751+ECE112 and also IA751 and 1A1 for control. August 13thResults for Starch plates- Add 5mL of iodine - 1A1 or IA751 : big zones of clearance - IA751 + ECE112 : no zones of clearance (photos), just small white points on colonies : the gene AmyE seems to be knocked out, transformation ok! - 1A1 + ECE112 : problem, soft agar melted... impossible to observe!
Transformation of IA751- We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171 - Spectrophotometer : blank made with medium A
- t0 = 120min - Follow the protocol of transformation - Plasmid miniprep of ECE153, 166, 171 (to make the transformation) - Nanodrop ( to add 0.5μg of DNA for transformation)
- Plate (with appropriate antibiotics)
Check our stock of ECE171- We made big stocks of ECE171, before keeping it, we wanted to check it, so we run double digest on a gel!! - Results : problem!! - 2 possible causes : Double digest was made " days before and kept in the freezer (possible degradation) + too much DNA on the gel - New double digest (from pellets in the freezer) + new gel - Results : ok! Glycerol stocks- for IA751 and IA771, add 100μL of culture and 500μL of glycerol (60%) Xylose experiment- To test the transformation of ECE153, we want to induce the promoter Pxyl, and we will have green fluorescence - Add 1mL of culture IA751 + ECE153 (from yesterday), 8mL of LB and 45μL of Spc50, 1mL of xylose (1g into 10mL) Plasmid miniprep for B.S.- We first tried to use the same protocol than for E.coli (Zyppy kit) for ECE166 transformed in IA751 - Nanodrop to see the result
- We have very low concentration of DNA, we will digest that plasmid tomorrow morning to see if it is ECE166, and if it does work, we will try to modify our protocole tomorrow Transformation of ECE188, 189, 190 in E.coli
In Preparation of Beta-galactosidase Assay (Promoter Assay PA)Biobrick Extraction: E0040
I13522
B0034
R0040
Transformation of competent TOP10 with the 4 biobricks above along with pUC19 control
August 14thResults of transformation
- Problem with our control, maybe we should check the resistance of competent cells (before transformation) BioBrick extraction for testing promoters and RBS in B.subtillisThe primers for inducible B.subtillis promoters have been ordered. Meanwhile, we would like to be able to compare the RBS and promoter strengths in E.coli and B.subtillis, using GFP fluorescence to quantify gene expression. We attempted to isolate 4 BioBricks: I13522 (GFP under constitutive promoter), E0040 (GFP only), R0040 (a promoter), & B0034 (an E.coli RBS). So far, only the former two, extracted from 2007 wells, grew after transformation. Single-colony PCR was used to test the transformants. Expected VF2-VR fragment sizes: I13522 - ~2.4 kb E0040 - 958 bp R0040 - 292 bp B0034 - 250 bp Transformation of vectors 188, 189, 190- Transformation of yesterday did not work! there was nothing on plates, even on the control plate - Try again, same protocol, 2μL of DNA, control : PUC9 - Plate on Amp100 for control and Amp100 + Cm5 for vectors Double digest of ECE166 (extracted from transformed Bacillus yesterday)- Transformation from 07/08 - 16μL of DNA, 2μL of Buffer EcoRI, 1μL of EcoRI (Biolab) and 1μL of HindIII - Incubator at 37°C for 35min, heat shock at 80°C for 5min - Run on a gel with 4μL of DNA
- Run on a new gel with 16μL of DNA
We are going to check this transformation with fluorescence too! Erythromycin plate- Erythromycin plate for the transformation 13/08/08 - IA771 (control, they should survive) - IA771 + ECE166 (non integration vector, so colonies should survive) - IA771 + ECE171 (integration vector but in another locus, should survive) - IA771 + 153 9integration vector, they should die if they are transformed) New stocks- LB stocks with antibiotic of ECE151, 153, 166 - LB stock of ECE153 with Spc50 (one from colonies from LB stock from 11/08 and one from 13/08 transformation plates) Check fluorescence with microscope
We diluted one colony from this plate into SDW and observed with microscope. Result : Bacillus is fluorescent! There are some bacteria which are brighter than others, but it is because it is not an integration vector. This transformation worked!!!
We added xylose, but we did not incubate our sample after that, so it did not work> We will try again tomorrow with a period of incubation> We will have to Plates from yesterday for Biobrick Extraction (PA)
Single Colony PCR of I13522 and E0040
First Row:
Second Row:
Result:
August 15thTransformation ECE188, 189, 190 into E.coli- Results : only our control plate grew!! - Maybe we did not add enough DNA, or there is another problem. We will e mail the guy from Bacillus center before trying again, because we don't have enough DNA. Plasmid miniprep ECE 151 (x2), ECE 153, ECE 166
Xylose Induction with ECE 153- 4 Tubes - No fluorescence! We have to think about the transformation of integration vectors into Bacillus.
Glycerol Stock Transformation- Spin down cells (competent) from 5/8 and 13/8 stocks - Remove liquid, resuspend in Medium B - Incubate for 60 mins at 37°C - Add ECE 166 into tubes - Incubate for 30 mins at 37°C - Plate out on CM 5 plates - 4 plates
Transforming ECE 188, 189, 190 (3rd try)- 2 tubes of chemically competent top 10 - Spin down, remove liquid - Resuspend cells in 100μL of CaCl2 50mM - 4 tubes with 50μL of cells
- Ice for 30 mins - 42°C for 2 mins - Ice for 2 mins - Incubate at 37°C for 2 hours - Plate out on Amp 100 (Neat) Biobrick E0040 and I13522 (PA)
August 16thResult from the days before
- Everything grew, we will try again to see if there is a problem with the antibiotic (and we will do a negative control with the strain IA751)
- We have a result from our 3 transformation in E.coli. To transform E.coli with these vectors we have to wait for 2 days before seeing cells!! We will check the vectors next week.
- Nothing! Problem with the storage of competent IA771??? August 18thTest Ery efficiencySince all Ery plates grew, we want to check the antibiotic stock. - New Ery plate of IA751 (they should die) - New LB stock (Ery5) with IA751 Single colony plates- New plates (Cm5 + Amp100) with single colony of ECE188 (from 14/08), ECE189 (from 13/08) and ECE190 (from 14/08) - Grow the same single colony into LB with antibiotic Transformation of glycerol stocks of B.S.The result from the last transformation of glycerol stocks was negative> We tried again with two different protocols.
- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B - Incubate about 1h15 - Add 0.6μg of ECE166 (because we managed to transform BS with this vector) - Incubate 1h30 - Plate on Cm5 plates (DNA less control and transformed cells)
- Spin the glycerol stock, throw out the supernatant (just keep about 100μL), add 0.5μL of Medium B - Add 0.6μg of ECE166 (because we managed to transform BS with this vector) - Incubate 30min - Plate on Cm5 plates (transformed cells)
PA with I13522, E0040 and B0034 Preparation
Gel:
Result:
August 19thResults from the transformation with glycerol stocksWe used two different glycerol stocks, one from 07/08 and one from 13/08. We also had 2 different protocols (cf 18/08 page). We tested our stocks on blank plates, cells are alive, the problem is to know if they are still competent or not.
- Stock from 07/08 : nothing on the control and nothing on the transformation plate. - Stock from 13/08 : nothing on the control and nothing on the transformation plate.
- Stock from 07/08 : nothing on the control and nothing on the transformation plate. - Stock from 13/08 : nothing on the control, and 1 colony on the transformed plate. We checked the fluorescence of bacteria into this colony (vector ECE166 with promoter and GFP). We have fluorescence, so our transformation is ok. The result of this confirm that our stock of competent cells in glycerol is still competnet since we managed to transform one colony. The problem is certainly not the storage, but our protocol when we use frozen competent cells> We need to improve the efficiency of this protocol. Test our Erythromycin stock- We put some colonies on a Ery 0.5 plate. They grow, but in a very small amount, so our antibiotic seems to be fine. - Cells in LB with antibiotic : no growth! So our antibiotic is ok, either we did not transform our cells and that is why everything grew on Ery plates, either there is a problem in our protocol for Ery test. Plasmid miniprep of ECE153, ECE166 and ECE171- Plasmid miniprep LB stocks from yesterday - Nanodrop
Transformation of IA771We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171. Spectrophotometer : blank made with medium A
There was a problem with our colonies. At t=80min, we inoculated again our tube of Medium A. - We used IA751 plate from 12/08/08, vectors ECE 153, 166, 171 - Spectrophotometer : blank made with medium A
 - Continue the protocol, add DNA (ECE153, 166, 171) - Plate on antibiotic plates
- 8 tubes : spin cells, throw away the supernatant, add 500μL of glycerol 60% - 8 tubes : spin cells, keep only 100μL of medium B, resuspend cells and add 500μL of glycerol 60% - Put them in the freezer at -80°C Dealing with R0040, R0062, S0168, I13522 for PA
Check I13522 biobrick DNA
Result:
August 20thCheck vector ECE190- Plasmid miniprep - Nanodrop - Double digest : 6μL of SDW, 8μL of DNA, 1μL of buffer, 1μL of XbaI and 1μL of PstI - Run on a gel  - Result : 2 bands (lane3, a little bit more than 3000b and a little bit more than 5000b) :ok! August 21stCheck ECE188- Plasmid miniprep - Double digest with XbaI and PstI - Gel (lane6)  - Result : nothing! not enough DNA August 22ndStock ECE112, 166, 153- Plasmid miniprep (2 tubes of ECE112, 2 tubes of ECE166 and 1 tube of ECE153) - Nanodrop Transformation of Bacillus
 - For glycerol stocks, resuspend cells into 100μL of medium B, add DNA and wait for 30min, end plate them August 23rdResults of transformation from 23/08/2008
August 26thXylose test
- Prepare 4 tubes with 10mL LB and Spc50, inoculate each tube with a colony from IA751 + ECE153 Starch plate- prepare some blank agar plates - add 1g of starch into 100mL of soft agar, mix, boil - put a thin layer of soft agar with starch - plate with IA751, IA771, IA751+ECE153 and IA751 (control plate, to see if we mixed some plates) - Incubate at 37°C Ery plate- Plate IA751, IA771, IA751 (control plate) and IA751+ECE153
August 27thXylose test
- Add 1mL of culture from yesterday, 1mL of xylose (0.1g/mL) and 8mL of LB and 45μL of Spc (do that for the 4 different colonies) - Incubate 5hours - Observation with microscope : some green task, but they do not really match with bacillus cells, not conclusive! Starch plate- Add 5mL of iodine solution on starch plate, wait for 1min, throw the liquid - Result
Ery test- IA771 alive and IA751 dead, confirmation that we did not mix plates (same conclusion than with starch plate, last line) Test glycerol stocks of competent bacillus- Pellet bacillus cells, add 100μL of prewarmed mediumB, add 0.6μg of DNA, incubate 30min at 37°C by shaking, plate
August 28thResults from Bacillus transformation with glycerol stocks
Test AmyE insertion
- 20μL of lysozyme, a few colonies for each - Cycle : 15min qt 37°C, 15min at 99°C, 1min at 4°C, 1min at 99°C and 1min at 4°C - for each PCR : 12μL of cells, 1μL of cells, 1μL of primer1, 1μL of primer2, 5μL of master mix - PCR (iGEM program) - run a gel 
August 29thStock of ECE112- Bulk up ECE112 (Amp100) rfom 10mL LB (28/08), take 1mL and add to 100mL of LB in big flask - Grow overnight in 37°C incubator with vigorous shaking - Aliquot in 10mL tubes, pellet and freeze cell pellets Transformation of glycerol stock
- Transfer into Eppendorf tubes and spin down for 30min -Remove glycerol and add medium B - Add ECE112 and ECE153, and one control tube - incubate for 30min in 37°C - Plate out ECE112 (Cm5), ECE153 (Spc50), DNA less (blank, Cm5, Spc50) September 1stResult for Bacillus transformation (27/08)
New Bacillus transformation (to make glycerol stocks)
September 2ndTransformation of Bacillus
- IA751 on Cm5 and Spc 50 (control DNA less plates) - IA751 + ECE112 (1μg of DNA) on Cm5 - IA751 + ECE153 (0.6μg of DNA) on Scp50 - IA771 on Cm5 (control) - IA771 + ECE166 on Cm5
Check ECE112 stock- Double digest with XbaI and EcoRI  - Gel : problem only one band (single digest???) September 3rdResult from bacillus transformation with fresh competent cells (02/09)
With 1μg of DNA, we manage to obtain colonies with ECE112. We will check our transformation with starch plates. Check ECE112 stock- Single and double digest with XbaI and EcoRI  - Gel
- Results : ok Test glycerol stocks (from 02/09) of competent bacillus
Starch plates- Use blank plates - Melt 100mL of Soft Agar, and add slowly 1g of starch - Mix, boil to sterilize - Pour on blank plate - Put 4 or 5 colonies on each plate
September 4thResult of Bacillus transformation (glycerol stock of competent cells)
We have no contamination in our glycerol stocks. However, the efficiency of glycerol stocks seems to be lower than these of fresh competent cells. Result of starch platesWe had 5mL of iodine solution, wait for 1min and observe. Controls (the small zone of clearing are contamination, we have not yet manage to find how to avoid them) Negative control : IA771, no zone of clearing  Positive control : IA751, big zones of clearing 
Colony PCR from bacillus subtilis coloniesWe want to check the size of the insert in the AmyE region(IA751) after transformation with an integration vector (to check if we have a single cross over or not). We are going to test our transformation of ECE112 and 153. We already tried this protocol, but our PCR did not work, so we tried again, with one sample of chromosomal DNA from bacillus to check if our transformation works. - add 20μL of 0.05mg/mL lysozyme into a PCR tube - add a few colonies into this tube until you obtain a dense solution. We test :
- 15min in 37°C, then 15min in 99°C, 1min in 4°C, 1min in 99°C, 1min in 4°C - For IA751, IA751 + ECE112, IA751 + ECE153, and bacillus chromosomal DNA, add 12μL of SDW, 5μL of MM, 1+1μL of primers (amyE primers), 1μL of the solution prepared befor with cells (or DNA) - PCR - Gel
 - Result : only chromosomal DNA worked. That means that our primers are ok, but we have to find a protocol for chromosomal DNA miniprep of bacillus to be able to PCR something into the genome of bacillus.
- Gel
 - Result : Lane4, we have something but too small.
September 5thStarch platesPrepare some starch plates. We want to test our transformation but we can not manage to PCR Bacillus. So we are going to try to extract chromosomal DNA from Bacillus. We make these starch plates to test it with colonies which have positive control with amylase test. - Prepare starch plates (you can find the protocol here) - Plate 4 different plates :
September 6thResults of starch platesWe had 5mL of iodine solution, wait for 1min and observe.
September 8thPrepare chromosomal DNA extraction from Bacillus- Grow 2 tubes into LB for
PCR AmyE from ECE112- Add 12μL of SDW, 5 μL of MM, 1μL + 1μL of each primers (for AmyE back and AmyE front) and 1μL of ECE112
September 9thChromosomal DNA extraction- ZR soil microbe DNA kit for
- Nanodrop
- Gel
- Only something for IA751, and ECE153 (pure plasmid)??? - New PCR with 4μL of chromosomal DNA and new PCR program - New gel
 - No insert in IA751 transformed with ECE153 and ECE112 (same size than control!) and however the amylase test is positive for this colony.... PCR AmyE again- 24μL of SDW, 10μL of MM, 2μL of each primers, 2μL of ECE112
- Very good result for amyE front, nothing for AmyE back September 10thCheck PCR for pSBINT1 vector- Gel
 - One band for rep, but not very clear, we should try again - for the other parts, we have a good band but the wrong size, these biobricks seem to be bad, we will order them from the MIT September 11thPCR for pBSINT1 biobrick vector
- Add 12μL of SDW, 5μL of MM, 1μL of rep fwd primer, 1μL of rep rev primer and 1μL of I732007 (program iGEM new)
- Add 9μL of SDW, 5μL of MM, 1μL of VF, 1μL VR and 4μL of each biobrick (program iGEM new)
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