Editing MBP fusion protein expression in CAG629 and CAG597 (section)

== <br>E. coli K12 CAG597 for periplasmic expression<br> ==

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A suspension of E. coli strain K12 CAG597 which has been grown in rich LB medium and brought to 50% glycerol. E. coli K12 CAG597 has rpoHam allele (sometimes referred to as htpRam) which encodes an amber mutant form of the sigma factor for the heat shock response. Grow by streaking out on an LB plate and incubating at 30°C.<br>Genotype F- lacZ(am) pho(am) tyrT[supC(ts)] trp(am) rpsL(StrR) rpoH(am)165 zhg::Tn10 mal(am)<br><br>Storage Temperature: -20°C<br>Antibiotic Resistance: str, tet

Growth Media: LB<br>Growth Temperature: 30 °C <br>

The strain has a temperature-sensitive amber supressor TyrT [supC(ts)] , so upon incubation at 37°C or 42°C the strain does not suppress the heat shock sigma factor RpoH. Thus it becomes deficient in expression of the heat shock proteins. Since a number of heat shock proteins affect proteolysis, this can increase the stability of foreign proteins expressed in E. coli. CAG597 is EcoK r+m+, so plasmids have to be modified (i.e., come from an m+ strain such as TB1, JM83, JM107, etc.) in order to get transformants; the transformation frequency is 10-100X down from other common strains used for recombinant DNA work<br>

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