Message+to+Carlos+at+Scripps+1-5-12


 * from: |||| Kurt Whittemore kurtwhittemore@gmail.com ||
 * to: |||| carlos@scripps.edu ||
 * date: |||| Thu, Jan 5, 2012 at 5:40 PM ||
 * subject: |||| pComb3XXS ||

Hi Carlos Barbas,

I am considering using your pComb3XXS vector to construct my phage antibody library. I just had a few concerns/questions. I see that this vector has the f1 ori. However, when I look at other phagemids, they have the f1 ori and ColE1 ori both. I'm not sure why this vector does not have the ColE1 ori. I don't understand the need for these oris clearly, but I thought that the ColE1 ori helped to make sure that the plasmid would be replicated when bacteria replicate.

Also, I see that this plasmid is designed for inserting the heavy chain and light chain variable genes separately, and these genes will be separated by a PelB Leader linker. However, before I started to consider using this plasmid, I have already constructed scFv genes in the form of heavy chain-linker-light chain where the linker is (Gly4Ser)3. Are there any potential problems with me just inserting my scFv the way they are now in place of the current heavy chain-linker-light chain in the pComb3XXS vector?

Thanks for any information you can offer me! Feel free to educate me about anything I am misunderstanding.

Best regards, Kurt Whittemore

Graduate Student Arizona State University BIODESIGN INSTITUTE Center for Innovations in Medicine 1001 S McALLISTER AVE TEMPE, AZ 85287