One gene in Bacillus subtilis should be deleted in my lab. Any confidity method or protocol or kit can be used? Is it same as operation for E. coli? What should I pay more attention?
It is pretty different from E. coli, since Bacillus is able to develop natural competence. When designing your construct make sure that you have approx. 500 bp homology regions left and right of the locus you want to delete. For Bacillus there are several protocols to make competent cells (you starve the cells in minimal medium with a certain amino acid composition, check cells microscopically for smaller cell bodies and increased motility) and in that stage recombinases are expressed for directing homologous DNA regions to their counterparts in the chromosome.
Good luck!
The Bacillus Genetic Stock Center has a complete library of single-gene knockouts in Bacillus subtilis 168. They are analogous to the Keio collection in E. coli. The knockout cassette specifies erythromycin resistance. It can be removed by a Cre-producing plasmid if desired, leaving a marker-free in-frame deletion for the locus. Please let me know if you have an interest or if you have a follow up question.
Can anyone recommend plasmids for knockout gene in Bacillus subtilis? Or a plasmid can be reconstructed?
I am actually trying to do the same currently and I came across multiple protocols using the splicing y overlap extension for the gene deletion then homologous recombination. I prefer the one attached as it is highly detailed and for this protocol you don't need ligation into a plasmid. You will have to order some strains though in order to amply the cassette they mention. Let me know if you have some questions (like where to order it). Hope this helps.
Hi Maimouna N Gadjaga,
Thanks for your protocol. It sounds very interesting. But most of all, as you mentioned, where to order TMO311 or how to get mazF?
The CRISPR-Cas9 genome editing technology has been applied to Bacillus subtilis. At the BGSC we have plasmid pJOE8999.1, as described in Altenbuchner 2016 (PMID: 27342565).
I'd also like to make it clear that we have in stock ~4000 single gene knockout strains of Bacillus subtilis 168, one for every non-essential gene in the genome. You can contact me for details if you are interested.
Article Editing of the Bacillus subtilis genome by the CRISPR-Cas9 system
Hi, Daniel R Zeigler,
Thanks for your KO collection file presenting how to use Cre. I want to know what is the origin of erythromycin resistance gene. I cloned the erythromycin resistance gene from plasmid pDG1663 including the promoter region, is it ok?
I construct the knockout cassette up_flank-lox71-erm-lox66-down_flank and tried to transform the cassette into Bacillus subtilis NCIB3610 by the method posted on BGSC. I got some transformants on the plates, but none of them were correctly knock out when varified by erm gene PCR. I don't know why.. Do you have any suggestions? Thanks a lot.
Hi Yan Frank,
my experience with the erythromycin resistance gene is that it extremely depends on the antibiotic concentration. I think i used something like 3 - 5 µg/ml. There are also protocols that use erythromycin in combination with lincosamid, which is said to help with selection. There are definitely better antibiotics (in my opinion) than erythromycin, for example spectinomycin (100 µg/ml) or kanamycin (10 µg/ml).
Good luck further on,
Flo
Dear Yan Frank,
The problem, I'm afraid, is that NCIB is only poorly competent due to the presence of a plasmid-borne inhibitory gene. Probably most of the colonies on your selection plates were resistant mutants, not transformants. There are two potential ways around this problem. (1) You could perform the strain construction in high competent Bacillus subtilis 168 and then transfer the knockout to NCIB3610 by SPP1 transduction. Alternatively, (2) instead of wild type NCIB3610 you could perform the constructions in a derivative, DK1042 (BGSC strain 3A38), in which the comI inhibitor has been inactivated. See Konkol MA, Blair KM, Kearns DB. J Bacteriol. 2013 Sep;195(18):4085-93 (PMID: 23836866) for a description of DK1042.
In either case, I would recommend selecting with erythromycin at 1 µg/ml. This selection level has been used successfully in literally hundreds of B. subtilis publications. Sometimes people use a combination of erythromycin (1 µg/ml) and lincomycin (25 µg/ml), since the erm gene confers resistance to both, and the combination of antibiotics reduces the number of spontaneous resistant mutants. In most cases using the combination isn't really necessary. And at any rate, your difficulties are caused by a low transformation frequency, not by spontaneous mutation.
Regards,
Daniel
Do you mean pJOE8999, Martin? If so, I have attached the sequence file in clone manager and fasta format.
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