I am working with RNA switches for tuning few genes for production of metabolites in Lactococcus. I have observed recombination events in my plasmids when I culture the respective strains for 24hours in a bioreactor. I am hypothesizing that the high cell density (almost 10 OD after a 24 hours bioreactor culture) causes recombination in between the switch-trigger pair sequences in the plasmids. I have verified this after sequencing them.
The reason could be that these switch-trigger sequences form very strong secondary structures and have reverse complementarity to each other, and thus recombine and omit out the sequences in between.
Now these switches are essential to my work. So I can not leave them.
Can you recommend me regarding what genes I can knock out in Lactococcus lactis for preventing these recombinations?
Thank you.
To prevent recombination events in your Lactococcus lactis strains, you can consider knocking out genes involved in homologous recombination. The genes involved in the homologous recombination process in Lactococcus lactis include recA, recB, recC, and recD. By disrupting these genes, it is possible to prevent homologous recombination and reduce the likelihood of recombination events in your plasmids.
Additionally, you can also consider reducing the growth rate of the bacteria during your culture phase, which may help to reduce the rate of recombination events. This can be achieved by adjusting the temperature, pH, or nutrient conditions, or by modifying the composition of the growth medium to reduce the rate of bacterial growth.
Hope it helps
By analogy to work in E. coli, knocking out RecA would abolish nearly all recombination. But those mutants do have pleiotropic effects that might impact your work. RecA mutants of E. coli grow more slowly and to lower density than wt mutants. You might want to review the literature on Lacococcus recA mutants before going that route.
Knocking out the recB/recC pathway would like have fewer pleiotropic effects and effectively reduces recombination although doesn't fully eliminate it.
Is the recombination event at a high enough level to impact your experiments or is it just something you detect and worry about? If it occurs but at a lower level it might be something you can just ignore.
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