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Contents 1 Third transformation of 'Bacillius subtilis 168' with Prrnb-GFP containing YneA 1.1 Aim 1.2 Materials and Protocol 1.3 Result 2 pH changing 3 Preparing medium for natto 4 Extreme Overnight cultures for Gibson

Third 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 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

pH changing

We brought hepes (which is similar to homopipes) to pH 7 then autoclaved it.

Preparing medium for natto

We prepared antibitoic ,medium for natto: 17.5g of broth in 1L, so 1.75 in 100ml

Extreme Overnight cultures for Gibson

1. G1 (T1) 1 Colony 1,
2. G1 (T2) 7 Colonies 2-6
3. G2 (T3) 2 Colonies 9,10
4. G2 (T4) 14 Colonies 11-24
5. G3 (T5) 3 Colonies 25-27
6. G3 (T6) 2 Colonies 28-29
7. G4 (T7) 4 Colonies 30-33
8. G4 (T8) 9 Colonies 34-42
9. G5 (T9) 0
10. G5 (T10) 2 Colonies 43-44