I knocked GFP into Bacillus at amyE locus but found results were interesting. Most successful transformants carrying Spectinomycin marker lose their ability to degrade starch. This was expected. However, in term of fluorescence, most of the transformants only have faint green glow under blue light but one of them is super bright even without inducers. I am confused which one is the norm of the right knock-in. Why is one much brighter than the others? The GFP plasmids were extracted from E. coli and use directly for transformation. They should not replicate in Bacillus.
In the colonies that were unable to degrade starch you are observing a faint green fluorescence, but in one colony you are observing an increase in GFP expression. It sounds to me the faint fluorescent expression is your norm. I'm not sure why it is brighter, but it could be an overlap of cells. If you were curious about that particular cell you could subculture it and observe for changes in fluorescence activity.
Thank you very much, Akos.
Actually, I used pDR111_GFP(Sp) in the paper you mentioned. I got lots of transformants that grew on Spectinomycin plates. When I put the plates (no IPTG) under blue light to excite fluorescence, I found a few transformants (about one in 100 ) was super bright. I did the transformation again and got exactly the same result. The GFP expression is always on without the inducer IPTG. I grew the transformant on other medium with/without Spc and they always had bright green fluorescence. It seems that some mutation occur at the promoter Phyperspank during transformation or that B. subtilis has some mechanism to constitutively express GFP independent of inducer. Or the plasmid has something unique.
Thank you again. I will compare the fluorescence of "normal transformant" with the "super bright" , both in the presence of IPTG. I agree with you that the mutation of promoter or lacI in the bright colonies might be a good explanation. I am also curious about that.
I got new data. The GFP production by normal transformants strongly depends on IPTG, while it is independent of IPTG by the super bright transformant. There is no difference in the fluorescence with/without IPTG for the "super bright". The super bright is about 60% more brighter than the normal even in the presence of IPTG. The data suggest that mutations might occur in GFP, lacI or the promoter. I will see whether I can sequence the inserted fragment. In terms of growth, there is no big difference in OD reading for both strains with/without IPTG.
@Jai, the insert do have Spectinomycin resistant gene but I never use the antibiotic when comparing GFP production. What do you mean "stop using Spectinomycin " ?
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