Hello All,
I am performing a Golden Gate assembly, and I've noticed that I've been seeing pale blue colonies alongside really dark blue colonies. My system is designed so that when the golden gate is accomplished, the complete lacZa fragment is eliminated, and white colonies should be correct clones. I went back into my backbone vector and saw an extra LacZa fragment (not a complete sequence) in my vector. Does anyone have any experience or opinion on whether this fragment may be enough to complement the LacZ gene in the bacteria?
The fragment sequence is SLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGE and the sequence is uploaded below if anyone is down to take a look.
I would appreciate any advice on this matter. Thank you!
I was looking at the plasmid you uploaded and there is an ATG start codon upstream of that fragment that's in frame which would lead to "MNQPHLLHDFLQVNSRGGPVPNSPYSESYYARSLAVVLQ...". In that case I think the ribosome binding site would be extremely weak, so if there's an active promoter upstream of it that would probably explain the weak positive.
It's also possible that an insert sequence can potentially leave LacZa in frame and fail to completely disrupt the lac+ phenotype though, depending on what the final sequence actually is.
Hey Alexandra, good looking out!
I found an upstream promoter (using a bacteria promoter predictor), did an RBS calculation simulation, and found that it's a pretty weak RBS that may be responsible for this phenomenon.
I would have to sequence and see what's going on fully obviously. I would be very curious what insert would cause this since my insert is meant to replace the entire LacZa fragment (the full version that I'm targeting with the Golden Gate).
I would add that you really don't need a promoter for weak expression on a multi copy plasmid. And that segment of LacZ should suffice for alpha complementation so long as you have an upstream start codon.
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