Many options are available. From your experience, what choice is the most efficient tool for gene knockout in bacteria, considering the time consumption, robustness of protocols, availability of reagents and plasmids.
I would say insertion of an antibiotic resistance cassette is a simple way to knock out a gene in bacteria
It all depends on what bacterial strain you are using. There are very different methods for different bacteria, depending upon how developed their genetic tools are.
Hi Daniel,
The best method reported so far is that commented by Kyle. Datsenko & Wanner 2000 reported that bacterial cells with mutation on the exonuclease component of RecBCD recombinant complex can integrate linear DNA. This method was originally developed in E. coli but variants of the system are applied to several model organisms (Bacillus subtilis). To knock out the gene is important to use an appropiate selection marker and antibiotic resistance genes are frequently used for this aim. I strongly recommend you to use CAT gene (Chloramphenicol Acetyl-Transferase = Chloramfenicol Resistance) because other genes such as that conferring Kanamycin resistance frequently produce a lot of false positive mutants because natural resistance of strains, then you turn to select with very high concentrations of Kanamycin. Good Luck!
Hi Daniel -
If you're working with E. coli or close relatives (Salmonella or other Enterobacteriaceae) the Datsenk/Wanner method with Lambda RED is the best. IF you're using other bacteria this method may not work (I've tried with some member of Acinetobacter and Pseudomonas and it doesn't work). For other bacteria, suicide vectors/allele exchange is a good way. Some bacteria, Legionella for example, are naturally competent and can be mutated with PCR products with an antibiotic cassette in place of the gene. Another beneficial tool is to add a counter-selectable marker, such as sacB (confers sensitivity to sucrose) that can be used to help remove the inserted antibiotic cassette after it's inserted where you want. Good luck
I think one of the biggest problem is to transfer DNA into recipient cells. One of the effective way is to use the conjugation system of E coli. This method is very common in genetic manipulation in Vibrio.
Looks like the tried and true classical methods have been mentioned. A newer approach to consider is the CRISPR-Cas9 system (http://www.ncbi.nlm.nih.gov/pubmed/23360965). The Marraffini lab CRISPR plasmids are available from Addgene (http://www.addgene.org/CRISPR/bacteria/). CRISPR-Cas9 system is more powerful, enabling targeted genome editing. The approaches already described would work best for simple knockouts however.
@ Norman,
Do you have any idea of how wide bacteria taxonomic range is good for using CRISPR-Cas system for gene knock-out / in? It seems that it works well for only some bacteria so far.
RNA-guided Cas9 from S. pyogenes, S. thermophilus and Neisseria meningitidis have been have been described. Fonfara et al. (http://www.ncbi.nlm.nih.gov/pubmed/24270795) showed that the phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems.
We use the lambda RED recombination system for E. coli and Salmonella (http://www.ncbi.nlm.nih.gov/pubmed/10829079).
Daniel - there is a pretty vast literature on generating gene knockouts in various Vibrio strains. I would review the literature for the strain that you are using (different Vibrio's are quite different and may not be generalizable).
I followed the exactly same protocol of wanner and i reached till the end but i am not getting any colony on the final kan knockout plate. I don`t know where I am lacking. I checked transformation again, induction process too but all steps are correct so can you give any suggestion regarding this.
i still cannot use the more effective kockout -gene method in Klebsiella pneumoniae. who can recommend some gene edit method about in this strain for me ? like CRISPR.@
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