Complementation in Staphylococcus aureus

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schwartz
schwartz's picture
Complementation in Staphylococcus aureus

Hi all,
I complemented a mutated gene in Staphylococcus aureus strain COL. The sequence is verified and everything seems to be right. The gene + flanking region is on a pCR2.1-TOPO + pSK265 shuttle vector. Total size is a little bit more than 8Kb.

The problem I have is that in phenotypic assays and sometimes in gene expression analysis, there's not enough complementation (only about 30-40% tops).

Any suggestions on why I am not getting good complementation? Any ideas of other plasmids that I can use as shuttle vectors will also be appreciated.

Thank you!

A Timmer
A Timmer's picture
Our lab uses a vector called

Our lab uses a vector called pDC123 which has a chloramphenicol resistance marker. There is a also a vector called pDCerm which is the same thing but with an erythromycin resistance marker. Some SA strains are naturally resistant to Erm so pDC123 might be better. Our lab has many papers using these complementation vectors, mostly in Group A Streptococcus but also in Staph.

http://nizetlab.ucsd.edu/Publications/

It is not uncommon to not get full complementation either. There could be polar effects of your mutation, but also expressing the gene on a plasmid means that it is regulated differently and there are more copies and there may be detrimental effects of overexpression. I think the most important thing is that the complemented strain is significantly different than the mutant, not necessarily the same as wild type.

schwartz
schwartz's picture
Thank you very much for your

Thank you very much for your response.