Design of an improved host platform for the over expression of recombinant proteins in Escherichia coli

The objective of this work was to design an improved host platform for recombinant protein expression in E. coli. The approach involves first to create a library of the E. coli genomic DNA in different expression vectors and screen for probable transcripts which may lead to slow growing colonies and also simultaneously over-expression of recombinant proteins. To observe its effect on host performance, these genes were knocked out from the E. coli genome. A CG2 strain has been created by knocking in vhb gene gene downstream of the acetate promoter and knocking down ribB gene in DH5α and transformed with Recombinant GFP cloned in pBAD33. E. coli DH5α (control strain), and strain CG2 were transformed with pBAD33-GFP and cultured in well controlled fed batch fermentations. The effect of three parameters was sought to be studied. The first was the effect of time post induction and thus samples were collected 0, 2, 4 and 6 hours post induction. The second parameter was the effect of host modification and hence both the control and the modified host (CG2) were studied at μ = 0.3 h-1. The third parameter was the effect of specific growth rate and thus the host (CG2) was run at μ = 0.3 h-1 and 0.6 h-1.