First transformation of 'Bacillius subtilis 168' containing Pmutin4 with Prrnb-GFP containing YneA

Aim

The aim of the experiment is to perform insert the plasmid Prrnb-GFP 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 antibiotic chloramphenicol and streptomycin, therefore those that have successful intergated will be selected with agar plates that contain these antibiotic. 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 locus. Thus those that have intergardted 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 Bacillus subtilis Note: Overnigth culture of 'B. subtilis 168' in MM competence medium was done the day before and the iodine test was performed the day after.

Result

Subtilin Immunity BioBrick

Aims

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

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 of the 16 minipreps are below:

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Hyperspankoid characterisation

PCR of the four parts was carried out again. Why? Restriction digests of the four parts using EcoR1. Gel of the 5 amplified parts 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