We have been struggling to get positive transformants when we used the commercial kit from TAKARA, Cat No: 3380, the included B. subtilis host strain is RIK1285. We have been following their protocol precisely which is available for online. We have further tried to manipulate the protocol by considering the recent improvements on B. subtilis expression, but still could not solve the transformation bottleneck. Did anyone already use this system or have any suggestion for the solution???
Thanks a lot for your answers,
Did the transformation of the E. coli HST08 go well? Does sequencing of the purified plasmids from the E. coli subcloning look correct? The Bacillus transformation from frozen stocks is not very efficient ever, maybe about 400 total colonies per ug, but freshly made competent bacillus can help. Keep in mind that most good E. coli transformations are several orders of magnitude more efficient than with this Bacillus.
I realize keeping my competent Staphylococcus aureus- a difficult to transform Gram positive bacteria like B. subtilis, as frozen stock for 16h , increases the transformation efficiency ;please bear in mind so many factors may create the transformation bottleneck ; growth phase when competent cells are made- it has to be precise, purity and concentration of the DNA (with a range of concentration the transformation frequency is linear) , selection . I dont know if you work with a control DNA(plasmid ) and what is the story with that angle ?
Recently we also bought the B. subtilis expression kit from Takara and are facing now the same problems. We tried different transformation protocols, but without success. This chat is more than two years old....have you solved the transformation problem by now?
Unfortunately, that system did not work at all for us. We had to use different strain WB800 and single vector system PMA0911 in order to express our proteins in bacillus. This time we were successful to see extracellular protein expression in the media. Hope that this will help you to decide about the current system.
Hi Ines,
we also use the WB800 strain and an acoABCL controlled vector for B. subtilis expressions up to 1 m3.
Best regards to Hohenheim :-)
Hey Wojtek,
nice to hear from you and thanks - also to Ugur Uzuner - for the advices! We didn´t give up yet, but for sure we will also try WB800 as host since you had good experiences with this system.
Kind regards from Hohenheim to Basel ;)
Hey Ines and Wojtek,
Could you please help me to get WB800? I am in desperate need of this strain. I can pay for shipping charges. Your help is greatly appreciated.
Please contact me at: [email protected]
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