Hello everyone,
I am struggling to obtain an overnight/culture for E.coli Stbl3 bacteria transformed with a plasmid containing the Cas9 enzyme and a GFP donor.
Although the bacteria grow well in a small volume (up to 20 ml), they fail in growing in larger volumes.
Should I assume somehow the Cas9 is expressed to some extent and is then toxic for the E.coli?
Thanks for any suggestions!
Hi there,
If it grows OK in small culture the issue is the scale up as the cell growth ability is normally exactly the same whatever the culture scale. Do you use exactly the same media for small and large cultures? There might also be something wrong with the glassware. Is it perfectly clean when using it?
Dominique Liger thanks for your reply.
Yes, everything was the same. I will incubate my pre/culture again and hope I have bacteria tomorrow!
Thanks again.
We have been working with a few cas9-containing plasmids in both E. coli and actinomycetes. So far we have not found it is toxic to these two kinds bacteria.
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