IGEM:Cambridge/2008/Notebook/Bacillus/2008/08/14

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Results 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.subtillis
The 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


 * Result : no bands! not enough DNA!

- Run on a new gel with 16μL of DNA

We are going to check this transformation with fluorescence too!
 * Result : " bands, one of about 2000b, and one of about 5000b> The big one seem to be too small...

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

 * ECE166 from the plate from 13/08

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!!!


 * ECE153

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)

 * Growth observed on Amp100 plates for I13522 and E0040 only but not for R0040, B0034 and pUC9 control
 * Re-plated overnight culture neat on Amp 75 plates and incubate at 37°C overnight

Single Colony PCR of I13522 and E0040

 * 5 colonies picked from each neat agar plate onto Amp75 plates
 * PCR following standard protocols
 * Run PCR product on 1.2% agarose gel at 70V which is later soaked in EtBr

First Row: Second Row:
 * Lane 2 - Hyperladder IV
 * Lane 3-7 - I13522 Colonies 1-5 PCR
 * Lane 9-13 - E0040 Colonies 1-5 PCR
 * Lane 2 - Hyperladder IV
 * Lane 3-6, 9 - R0010 Colonies 1-5 PCR

Result:
 * Expected size of band: I13522 - 2375bp; E0040 - 958bp; R0010 - 292bp
 * Picked colonies 4 and 5 for E0040 into 10ml LB with Amp100 and incubate overnight at 37°C as the band corresponds to about 958bp
 * Band of R0010 colonies 3 and 5 correspond to 292bp and will incubate in LB tomorrow when colonies have grown on agar plates


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