HELP : Problem in Subcloning

rgupta at imtech.ernet.in rgupta at imtech.ernet.in
Thu Sep 7 10:52:07 EST 1995


Dear Netters :

I am facing a peculiar problem during subcloning. Whenever I try to ligate a
 stretch of DNA coding for around 100 amino acids at the 3'-end of the gene
 coding for Streptokinase (SK), I always get transformants which harbour all
 different kinds of plasmid species but not the desired one, i.e., the fusion
 product of the two genes.
The different kinds of plasmid species include :
i) Vector self-ligated - which, of course, is possible if any of the
 restriction digestions is incomplete.
ii) Low molecular weight species - in which some portions of the vector and/or
 SK gene have been deleted. Generally one particular molecular weight species
 predominates. On analysing these deleted mutants some of them were found to
 contain the fusion of the two genes, but as  a result of internal deletions
 these are unable to express the protein. To confirm that these low molecular
 weight species were not of pre-transformation origin _ the ligation product
 (before transformation) was run on agarose gel which did not show any low
 molecular weight bands.
The details of the experiment are as follows :
The expression system is E.coli and we have tried 'Sure' (Stratagene) and 'AG1'
 strains. The expression vector is a derivative of pBR322 where tetracycline
 resistance gene has been replaced with tac promoter, transcription terminators
 and laq Iq gene. It contains SK gene downstream of the promoter. The SK gene
 has secretory signal and is stably expressed and secreted out. This is a
 relatively hydrophobic protein. The total size of the vector is around 8 kb.
 The product of the gene which I am trying to fuse with SK  contains four
 disulfide bridges. I am using directional cloning with enzymes BamH1 and
 BsrG1 and electroporation for transformation.
In control experiments, transformation of undigested vector or transformation
 after single digestion and ligation gives right kind of transformants.
I have successfully done this experiment in pBluescript (Stratagene) vector
 where the expression is very less.
In a separate isolated experiment , I was trying to introduce a stop codon in
 the middle of the SK gene. In a 'cassette' of this region of SK gene cloned
 in M13, I had introduced a stop codon using site-directed mutagenesis. When I
 tried to put back this mutant cassette into wild type gene in the above
 expression vector, the results were the same as above. This experiment was
 also successful in the pBluescript vector.
I am not able to understand the reason . Is this problem because of the
 'nature' of the vector (e.g. 8kb size) which does not like fiddling ! Or I am
 missing some important point. I will appreciate any suggestions from you.
Thanking you,
Rajesh Kumar
	PhD

rgupta at imtech.ernet.in

Institute of Microbial Technology 
Sector 39 A, Chandigarh-160014
India



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