E-mail+to+Kathy+about+this+phage+display+kit+on+1-3-12

This is kind of a long e-mail so we could meet and I could verbally describe it to you if you want. Nevertheless, all the information is here if you want to read it. Also, when do you want to meet to discuss the cDNA library glycerol stock? I understand what you say about how the glycerol stock is not the best form of storage. However, I don't think it is possible to do what we want to do without it. I can give my argument when we meet. The rest of the e-mail about phage is below.

I've looked a little more into the PhD Phage Display Cloning System from NEB which uses the M13KE vector and is usually advertised as a method for displaying peptides <50 aa. Our scFv are 250aa.

The first thing I looked at was how the position of the peptide insert in the PhD Phage Display System compares with the position of the scFv in the PCANTAB5E plasmid. They are quite similar. The insert starts at 106 bp after the start of the pIII gene (1376 bp) in the PCANTAB5E vector. The insert starts at 32 bp after the start of the pIII gene (1274 bp) in the M13KE vector. A BLAST of the 2 different pIIIs reveals that the two genes are mostly identical but slightly different. 91% of the two genes is exactly identical, but then there are a few differences the gene in the two different plasmids at the beginning of the pIII gene sequence. I would think these differences are minor.

The next thing I looked at was whether anyone else has used the M13KE vector for an scFv phage antibody library. I ended up finding one patent by two Japanese inventor in which they do use this M13KE vector from NEB to produce an scFv phage. http://www.freepatentsonline.com/y2008/0206784.html I know that the infectivity of the virus decreases the larger the insert is so I wasn't sure if the M13KE would work well for scFv, but they seem to have done it. I sent them an e-mail last week asking them if it worked okay for them, but they are in Japan and it is the holidays so I may not get a response.

There are also a few more issues to consider. M13KE is a phage rather than a phagemid vector. All 5 copies of pIII will be fused to the cloned peptide. The plasmid has no antibiotic resistance. Also no helper phage is required. Perhaps all of these facts are okay for my purposes as long as the phage can still be infective with the scFv. Also, if I were to use this system, I guess I would not be using the M13cp-CT helper plasmid we obtained from Texas.

Finally, the M13KE vector has different restriction sites than SfiI and NotI which supposedly occur rarely in mammalian genes. The M13KE vector contains Acc65I-KpnI and EagI at the insert site. Therefore, we would either need to change the restriction sites on my scFv genes from the PCRs or we would need to change the restriction sites of the vector.

So from all of this information I think that we could use this M13KE vector for my phage antibody library. However, there's a very good possibility that I could be misunderstanding something with how the phage will be produced and whether or not we can do without helper phage and everything.