Has anyone successfully used pKD46 as a lambda Red recombinase system for gene deletion in Bacillus subtilis? If so, what challenges or modifications were required to achieve efficient recombination?
Yes pKD46 can be used for gene deletion in Bacillus subtilis, it often requires adaptations in terms of promoter choice, expression conditions, and transformation protocols. If you plan to use pKD46 for recombination in B. subtilis, expect to make modifications to the system, especially in terms of optimizing the induction and recombination conditions.
Katashkina, J.I., Hara, Y., Golubeva, L.I. et al. Use of the λ Red-recombineering method for genetic engineering of Pantoea ananatis. BMC Molecular Biol 10, 34 (2009). https://doi.org/ 10.1186/1471-2199-10-34
While the λ Red recombinase system, including plasmid pKD46, is primarily optimized for use in Escherichia coli, there have been studies exploring its application in other bacterial species, such as Pantoea ananatis. In the study, the authors successfully employed the λ Red system to introduce chromosomal modifications in P. ananatis, utilizing PCR-generated DNA fragments with as short as 40 base pair flanking homologies.
https://cafgroup.lbl.gov/protocols/general-molecular-biology/genomic-gene-deletion-with-lambda-red/protocol-for-gene-knockout
Researchers interested in using the λ Red system in B. subtilis may adapt protocols, such as modifying the promoter driving the expression of the recombinase genes, optimizing induction conditions, and ensuring compatibility with B. subtilis's native recombination machinery.
Strongly disagreeing with Nicolas Poirier . Pantoea is not a comparable genus to Bacillus. Pantoea is in the same order (Enterobacteriales) as E. coli, the organism which pKD46 was designed for. The genus Bacillus is not even in the same kingdom.
Here are the problems with getting this plasmid to work in Bacillus:
1. The pKD46 origin of replication will not function in B. subtilis.
2. The promoter to drive the lambda recombinase (E. coli arabinose promoter) will not function in B. subtilis.
3. The ampicillin resistance gene will not express in B. subtilis, but even if you gave it a Bacillus promoter it would still be non-functional because B. subtilis will not recognize the signal peptide to secrete the beta-lactamase where it needs to go.
4. The Gam protein which improves the efficiency of the lambda recombinase system by inhibiting the Rec pathway is known to be poorly functional when interacting with Rec proteins distally related to E. coli.
5. Even if you switched out all the promoters and the origin of replication to ones that actually work in B. subtilis, the E. coli ribosome binding sites and codon usage would result in poor expression.
You're much better off researching tried and true methods for genetically manipulating B. subtilis than wasting your time trying to adapt pKD46, which would essentially require changing almost every part of it.
Alexandra Johnson I agree with you that it is not possible to directly use the pKD46 plasmid in Bacillus due to compatibility issues with its replication system and recombination mechanism.
There are alternative strategies and plasmids designed specifically for genome manipulation in Bacillus. If you wish to perform homologous recombination or gene editing in Bacillus, using species-specific tools, such as pMUTIN plasmids or the pG+ host system, can be an effective approach.
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