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

Gibson for rocF

• Promoter 136bp - 0.7 microl
• rocF 1 246bp - 1.2 microl
• rocF 2 597bp - 1.9 microl
• rocF 3 125bp - 0.4 microl
• DTERM 116bp - 0.8microl

Total: 1190bp

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 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 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.