From 2012.igem.org
Team:Technion/4_April_2012
Ilya
Evgeni found a few sources for the T7 RNA polymerase gene. I managed to find a source for the SP6 RNA polymerase in some papers that tested SP6 promoters in vivo. The T3 RNA polymerase gene is hard to find though. I found the original paper in which the gene was sequenced and Sharon sent an email to the author. However, the paper is very old and the author is doing different research by now. Worst case scenario, we will need to order the sequence of the gene.
I also looked into papers which tested the effect of mutations on promoter strength for our three RNA polymerases. If we will want to fine tune the promoter strength, we might get some ideas from these papers.
Roee talked to me today and asked me to look into the E. coli strain we will be using. He suggested we use three of the following inducers: tetracycline, IPTG, AHL or arabinose. In order to do so, the strain we choose has to have endogenous expression of three of the following transcription factors: TetR, LacI, LaxR or AraC. To get me started he sent me a paper which used the strain DH5alphaZ1. That strain had a natural expression of AraC and high expression of LacI and TetR from the Z1 cassette which was integrated into it's chromosome.
I did some more research and found the strain MGZ1X expresses LacI, TetR, AraC, and LuxR. However, LuxR is expressed from a plasmid. Roee told me we are trying to avoid the extra plasmid and that we need to find an actual supplier for the chosen strain. So I continued my search, determined that the next set of results I get will answer all our needs.
I found the strain λALT which had pZS4-λcI-araC-lacI-tetR-Int integrated into the attB site of DH10B. However, the cI gene can be a problem in our project because it represses the lytic cycle of the lambda phage. Another strain is MG1655 Z1 which is similar DH5alphaZ1.
In case we can't get the strains from the authors of the papers, I found a commercial source for DH5alphaZ1: http://www.expressys.com/main_strains.html
Roee suggested I look into some databases. I found http://cgsc.biology.yale.edu/index.php andhttp://www.atcc.org/ but they weren't very helpful. However, the following list from the parts registry pointed me in the right direction for the commercial strains:http://openwetware.org/wiki/E._coli_genotypes#STBL4
I learned about the different mutations and how they are marked in the strain description. The list also had names of companies and strains that answered our needs. I also learned that any E. coliderived from the K12 strain naturally expresses AraC. So all I needed to find now is a commercial strain derived from K12 that was also expressing LacI and TetR (this was the most common combination).
Armed with this new knowledge I was ready to hit the catalogs of the big companies. Here is what I found:
I looked through the NEB strains catalog and found two K12 strains that have LacIq and TetR:
KS1000 - http://www.neb.com/nebecomm/products/productE4128.asp
ER2738 - http://www.neb.com/nebecomm/products/productE4104.asp
I looked in the invitrogen catalog aswell and found the following:
OmniMAX™ 2 T1 - http://products.invitrogen.com/ivgn/product/C854003
Stbl4 - https://products.invitrogen.com/ivgn/product/11635018?ICID=search-product
They have LacIq and tn10(TetR). Doesn't specify mutations in ara so I think it has AraC too.
Hopefully, one of these strains is what we are looking for :)
Inbal
Asaf
Hila
Lior
Noa
Evgeni
Shahar
Rachel
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