Hello,
I recently bought a pre-engineered e.coli Bl21 containing a pje202 plasmid. Yet I have to grow it on lb-ampicillin-agar to prevent plasmid loss, but I don't want to. I wondered, if there is a (for a student do-able) possibility to integrate the cyclic plasmid dna directly into the bacteria genome??
I have also researched into transduction, but that seems to be just too complicated for me. I have U3 coliphages, but that seem to be rather useless for integration of plasmid dna, right?
So any ideas, what's the easiest way to integrate plasmid dna into bacteria genome??
Hello Leonhardt, the issue to successful integration of plasmid into bacterial genome is heavily organism-dependent. In some, single homologous cross overs suffice but in most, double cross overs are required and this can be a problem. However, the problem can be overcome using a counter-selection marker located on the backbone of the plasmid. A major uphill task in this case is identifying a suitable counter-selection marker and the appropriate conditions that permit its use. But Heap et al. (2012) has a fantastic way out; See also Gu et al. (2015). They have been very useful to me.
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