expression under fungal promoter in bacteria (?) - (Oct/23/2005 )
I have serious problems with cloning a gene of interest into a fungal expression vector. Ligation seems to be OK but i get no bacterial colonies carrying ligation product. All i get are empty vectors.
I repeated it over and over again and still nothing.
Gene is to be cloned in-frame with an A.nidulans gpd promoter and GFP.
Is it possible that its expression (under gpd promoter) occurs in E.coli ?? The protein might be toxic for bacteria and that's why i get no clones?
pls pls help me
thnx a lot
Well.. it is possible
What is your cloning strategy (enzymes used, etc.)?
Maybe try this technique instead:
It cuts out the annoying cloning steps associated with building transformation vectors for fungi.
A. nidulans - dear to my heart...
I've done a lot of expression cloning in Aspergillus and haven't yet encountered a problem with expression issues in E.coli. Of course this could be the constructs I've used but here's a tip.
How big is your gene?
Empty vector may be the result of a more basic cloning problem. Are you using sticky ends? If not you probably should, especially if the size of the gene is above 1kb and since you require the correct orientation.
And of course, make sure all fragments are clean and the ligase is good.