Hello!
I try to make competent cell using Bacillus subtilis ISW1214 labatory strain. I did transformation pUC19 vector to confirm my competent cell efficiency. However, It didn't work well... Here is my protocol Please give adivce!
1. Inoculate a single colony in 5 ml TSB Grow overnight at 37 ℃.
2. Inoculate use 1.5 mL to inoculate 150 mL TSB
3. incuvating until OD600 of 0.5(3~4h) with shaking
4. Centrifuge 2,000 rpm, 10 min to cell down the 150 mL TSB
5. Washing 3 times using cold 0.5M sucrose
6. Discard supernatant and gently resuspend on 1 ml cold 0.5M sucrose
7. Dispence in 1.7 ml e/p tube(100 µl). Freeze in - 70 ℃
All of the process is carried on the ice and after washing, I did resuspend the cell down product using pipeting
To make B. subtilis (168) competent I use the following protocol:
To make B. subtilis competent for transformation, a 5ml culture was grown in minimal media overnight. The next day 300 μl of the culture was used to inoculate 5 ml fresh minimal media and was incubated with shaking at 37°C for 3 hours. At this point, 5 ml starvation medium was added and the culture incubated for a further 2 hours at 37°C. 400 μl of the now competent cells were used immediately for transformation.
400 μl of competent cells were mixed with 5 μl plasmid/ligation product or 10 μl genomic DNA. The mixtures were incubated at 37°C for 1 hour before plating agar containing the appropriate antibiotics.
Minimal media:
- 10 ml SMM ( Spizizen minimal media)
- 0.125 ml 40% glucose
- 0.1 ml tryptophan solution
- 0.06 ml 1 M MgSO4
- 0.01 ml 20% casamino acids
- 0.005 ml 2.2mg/ml ferric ammonium citrate
Starvation:
- 10 ml SMM
- 0.125 ml 40% glucose
- 0.06 1 M MgSO4
Also pUC19 is an E coli plasmid so probably isn't replicated in Bacillus
pUC19's origin definitely doesn't replicate in Bacillus. The origin actually gets used as a suicide plasmid for species outside the order Enterobacteriales because it replicates to a high number in E. coli but usually replicate at all doesn't in more distantly related species, it doesn't even replicate in Pseudomonas which is much more closely related to E. coli than B. subtilis is. You'll have to test transformation efficiency with a shuttle plasmid (plasmid with E. coli origin and a separate Bacillus origin), a very broad host range plasmid (can replicate in a huge variety of organisms) or a plasmid that's been directed extracted from Bacillus.
The Main problem in your method is the plasmid type that you have used for transformation. Protocol for competent cell seems alright, and negative response is due to the fact that pUC19 does not replicate in Bs.
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