Do you have any particular strategy? Do you use higher temperature to cultivate bacteria (in my case this is Burkholderia and plasmid is a megaplasmid of 0.5 Mb) to get rid of non transformed plasmid copies? Thank you in advance for all suggestions.
Hi Agnieszka,
Without much background information, I can suggest you to check about the use of short interfering RNAs. I think if somebody know in depth about the siRNA, can improvise my answer further, so that its useful for you.
Hi Agnieszka, Unfortunately, I don't think siRNA is the way to go since this approach doesn't work in bacteria (due to lack of RISC/DICER mechanisms). I'm not very familiar with genetic tools available for Burkholderia. Are you able to isolate strains that have lost this plasmid (or cure in someway) or is it essential? If so, could you engineer a plasmid with gene of interest knocked out and re-introduce into the cured strain? This sounds like a problem that Borrelia researchers run into, since that bug has multiple linear and circular plasmids. I will talk to a Borrelia researcher down the hall from me and see if I can get additional insights.
Hi Kyle
Thank you for your help. Unfortunately, this megaplasmid harbors several genes of interest for symbiotic behaviour of bacterium so it is hard to imagine the construction of a new hybrid plasmid.
In fact, when I do deletion of the gene or insertion of 2 kb into the gene, PCR verification gives always a picture of the presence of both situations : WT gene and a gene with the mutation (deletion or insertion), even after several stricking of the bacteria on a selective medium (always starting with a single colony - so it is not a mix of WT bacteria with a mutant). That is why I think that I have this megaplasmid in at least two copies and I mutagenise only one copy of the plasmid.
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