IGEM:Cambridge/2008/Notebook/Bacillus/2008/09/04

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Result of Bacillus transformation (glycerol stock of competent cells)

strain antibiotic added vector observation conclusion
IA751 none none confluent colonies cells alive
IA751 Spc50 none nothing no contamination
IA751 Spc50 ECE153 15 ok, efficiency 25 colonies/μg of DNA
IA771 none none confluent colonies cells alive
IA771 Cm5 none nothing no contamination
IA771 Cm5 ECE166 15 ok, efficiency 5 colonies/μg of DNA

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 plates

We 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

strain vector photo observation conclusion
IA751 none
big zones of clearing for each colony ok
IA771 none
no zone of clearing for each colony ok
IA751 ECE112/none (29/08)
clearing zones for control, so no trnasformation, it is maybe E.coli, 1 clearing zone out of 3 colonies, it is certainly contamination, but that means that some colonies are transformed ok
IA751 ECE112 (02/09)
1 zone of clearing out of 4 colonies some colonies are not transformed and have a resistance to Cm5 (contamination?)
IA751 ECE153 (28/08)
no zone of clearing 5 transformed colonies
IA751 ECE153 (02/09)
2 zone of clearing out of 5 colonies some colonies are not transformed and have a resistance to Spc50 (contamination?)

Colony PCR from bacillus subtilis colonies

We 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 :

  • IA751
  • IA751 + ECE153 (29/08, 02/09, 03/09)
  • IA751 + ECE112 (02/09)

- 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

  • Lane1 : hyperladderI
  • Lane2 : IA751 + ECE153 (29/08)
  • Lane3 : IA751 + ECE153 (02/09)
  • Lane4 : IA751 + ECE153 (03/09)
  • Lane5 : IA751 + ECE112 (02/09)
  • Lane6 : IA751
  • Lane7 : Chromosomal DNA

- 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.

  • New attempt without the lyse process (only protocol of single colony PCR, like for E.coli)

- Gel

  • Lane1 : hyperladderI
  • Lane2 : IA751 + ECE153 (29/08)
  • Lane3 : IA751 + ECE153 (02/09)
  • Lane4 : IA751 + ECE153 (03/09)
  • Lane5 : IA751 + ECE112 (02/09)
  • Lane6 : IA751
  • Lane7 : Chromosomal DNA

- Result : Lane4, we have something but too small.



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