First transformation of B. subtilis 168 containing pMutin4 with pGFPrrnB containing yneA

Aim

The aim of the experiment is to perform insert the plasmid pGFP-rrnB containing yneA which have been ligated eariler into the chromosome of Bacillus subtilis 168. B. subtilis containing the intergated vector will be resistance to both the antibiotics chloramphenicol and streptomycin, therefore those that have successful intergated will be selected with agar plates that contain both the antibiotics. The second step will be to identify those colones that have the plasmid intergated at the corerct position in the chromosome, which is the amylase gene locus. Thus those that have intergrated at the wrong position will not be able to break down starch, which can be tested with the iodine test.

Materials and Protocol

Please refer to: Transformation of B. subtilis Note: Overnight culture of B. subtilis 168 in MM competence medium was done the day before and the iodine test was performed the day after.

Result

The transformation was unsuccessful.

Subtilin Immunity BioBrick

Aims

The aim of this experiment is to perform minipreps of the 16 overnight cultures that were left overnight from yesterday for the Subtilin Immunity BioBrick.

Methods

The Qiagen Miniprep protocol was used for each of the 16 cultures, followed by the NanoDrop Spectrophotometer protocol.

Results, Discussion and Conclusion

The nanodrop spectrophotometer results for the 16 minipreps for Subtilin Immunity (and rocF?) are below (units: ng/ml):

1. 76.5
2. 84.6
3. 61.5
4. 19.5
5. 89.9
6. 87.6
7. 6.3
8. 69.5
9. 81.3
10. 74.4
11. 68.5
12. 70.2
13. 83.5
14. 77.6
15. 73.3
16. 63.0

Hyperspankoid characterisation

• PCR reactions of the four parts that were carried out yesterday were carried out again today but with a control as well. Why?
• Restriction digests of the four parts using EcoR1.
• Gel of the 5 amplified sequences with a control.

Materials and Protocol

Please refer to Phusion PCR, Restriction Digest and Gel Electrophoresis for Phusion PCR, Restriction Digest and Gel electrophoresis protocol.

Results

GEL IMAGE

Results, Discussion and Conclusion

... Gel extraction will be carried out tomorrow.

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