In my undergraduate research project, I require an integrative plasmid containing GFP with an antibiotic resistance that can't be kanamycin. In our lab, all of our integrative-GFP-containing plasmids are resistant to kanamycin, so we decided to switch it's resistance using Gibson Assembly. The plasmid in question is called pL5-GFP and the KanR version (vector) was amplified by "reverse" PCR. The insert (HygR) was also amplified by PCR with 15 complimentary base pairs, and they were combined using the Gibson Assembly kit. The confirmatory PCR's (using the same primers as before) were positive, but after transformation, the mycobacteria recovered wasn't green. Then, we decided to do an restriction analysis, whose agarose gel showed inconclusive bands, which suggest that the new pL5-GFP-HygR is smaller than the KanR by approximately 3kb. I don't know what to improve in the case of starting over or what other techniques I could use. Any insights are helpful. Thank you.
ps. pL5-GFP-KanR successfully turns mycobacteria green.
Without the information on the restriction sites used in the analytical digest, it's hard to tell what's really going on.
I don't have any experience with mycobacteria but a lot with Gibson cloning.
The suggestion which most likely gets you the desired plasmid would be a short detour by doing the cloning in E. coli (if the ori is compatible), save the time and reagents doing any test PCRs on the in-vitro Gibson product, the Gibson flanks can just transiently anneal and give you a correct band even without proper ligation. Instead, I would transform the reaction and do colony PCRs with the resulting resistant colonies. Then, do mini preps from the ones which look good and send 4+ clones to sequencing (with the low prices for sequencing, it's almost never worth anymore to do additional restriction analyses before).
Another improvement would be using longer Gibson flanks (I usually do 20-nt flanks, longer doesn't hurt in my experience but doesn't improve the efficiency, but I've seen some differences between 15- and 20-nt flanks).
And lastly, just checking that you're using a proof-reading polymerase which doesn't produce overhangs for the PCRs to make the fragments which go into the Gibson (or have a kit which allows a few mismatches).
I hope these suggestions help and you'll get your plasmid and green mycobacteria soon.
Christian Renicke, thank you for your answer!
Actually, the cloning process was made in E. coli, and the colony PCRs were positive, but as soon as we transformed in mycobacteria, we saw that the plasmid wasn't functioning as we thought because the colonies we obtained were white. That's when we decided to try restriction analysis and had those "weird" bands. The enzymes used were EcoRV and SpeI, and I'll attach the maps containing the sites, in case you have any other insights.
Thank you for all your suggestions, and we will definitely increase the number of nucleotides in the flank when we repeat it. I'll also double check our polymerase.
Overall, thanks again for your help!
You shouldn't rely on colony PCR alone to confirm that you have the right clone. Do a mini prep and do some restriction analysis. This is because you might have a mixed plasmid prep and if even a small percent are what you want, then you would get a positive signal by PCR but when transforming the majority would be the wrong thing. So go back and analyze the original plasmid more carefully first.
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