Following up on my earlier experiment,
On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:
-- I have scrapped off "mutant" bacteria from the strep plate and spread it onto a fresh strep plate and a normal plate.
I have scrapped off wild type bacteria from the strep plate and spread it onto a fresh strep plate and a normal plate.
The first picture below shows the plates just after a spread the bacteria on them.
The second picture is of the same two plates after 2 days of incubation at around 37 C.
I guess there is growth on all 4 colonies, no real difference between strep plate and normal plate. No real difference between "mutant" and wild type on strep plate.
So i cannot conclude with this that i have successfully CRIPSR'ed that bacteria. But i cannot rule out that i have edited it either.
Puzzling to see growth of wild type on strep plate. Maybe there is no streptomycin finally on that plate. Or as it was earlier proposed, i might have deactivated the strep when preparing the LB gel. I did boil it a few seconds in the microwave.
I'll try to find a cheap place to sequence these 2 colonies, just to get the final call.
On Thursday, 22 December 2016 20:20:47 UTC+1, Hugues wrote:
Hi guys,i think i have successfully CRISPRed an E. Coli bacteria,I have used the kit sold here:I followed the protocol here:The gene that was targeted is Escherichia coli 30S ribosomal subunit protein S12 (rpsL) gene. One base pair was changed to render the bacteria resistant to streptomycin.The picture attached shows my result. That plate contains a gel with streptomycin. The top part shows growth on the CRISPRed strain, the bottom part shows not growth on the normal strain.What you guys think ?I'm thinking of having the DNA of that gene sequenced, just to check if the edit really happened only where expected.
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