Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Loss of promoter during ligation - (Aug/19/2011 )

I've been having trouble ligating a sequence into a pQE vector from Qiagen. Out of 28 colonies in my first trial I found one that had an insert, however the insert ended up being some weird genomic sequence. On my second try I used a phosphatase and found 3 colonies with inserts. All three had the correct insert after sequencing, but the -10 promotor region and the second lac operon sequence were missing upstream of the insert in all three. The vector on my first try maintained the correct promotor/lac operon sequence. I'm currently sequencing another set of colonies.

Has anyone ever had this problem during ligation? I'm wondering if the bacteria does not like making my protein and somehow got rid of the promoter sequence. It's strange that the ligated region is completely fine but the upstream area has a huge deletion. Any thoughts are appreciated.


Indeed, that's a weird phenomenon. Two things come to my mind:

1) Not a very clean plasmid prep. Could you have got some trace nucleases which would kick in after digestion, in such a case most probably you wouldn't see the restriction site you used on the insert side as well.
2) If you are using a second hand commercial vector this might have had some deletions i.e. are you sure of the vector sequence?

If you can eliminate the above 2 than indeed your protein might be toxic and the fact that you use pQE with T5 promoter wouldn't help due to higher leaky expression. What you could try in such a case is to consider much tighter expression by cloning your protein in a T7 based promoter vector and a expressing in strain with pLysS like Rosetta. Additionally, given that you are expressing an enzyme you could coexpress with another that would counteract its activity e.g. target kinase and coexpression of phosphatase.



Thanks for the ideas. I did some sequencing and it's not #2. Since RE sites are intact and insert is sequencing well I'm not sure if it is #1, but I can try re-prepping the plasmid.

I'm really leaning toward toxicity. I'm expressing this in DH5a in order to amplify the DNA, but a line that overproduces the lac repressor might work better.