First transformation of 'Bacillius subtilis 168' 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 integrated vector will be resistance to both the antibiotic chloramphenicol and streptomycin, therefore those that have successful integrated will be selected with agar plates that contain these antibiotic. The second step will be to identify those colones that have the plasmid integrated at the corerct position in the chromosome, which is the amylase locus. Thus those that have integrated 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.

Results

Unfortunately our colonies had halos. Although the insert has integrated, the colonies have antibiotic restistance, the insert has not integrated at the amyE locus.

However when we checked the colonies under a microscope the cells were filamentous due to integration elsewhere on the Bacillus subtilis 168 chromosome perhaps at the native yneA locus.

Filamentous cells

Filamentous cells showing GFP signal

Normal Bacillus subtilis 168