Loss of promoter during ligation
Posted 19 August 2011 - 08:03 AM
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.
Posted 19 August 2011 - 12:48 PM
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.
Posted 22 August 2011 - 06:22 AM
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.