E. coli K-12 is well established as a preferred host for commercial production of recombinant DNA products including enzymes, vaccines, metabolites and DNA itself. The complete genome sequence of the E. coli bacterium has now been determined and it shows the presence of many genes that are clearly unnecessary for production purposes and which are arguably detrimental. These include genes encoding phage remnants, adhesins, toxins and ORFs in pathogenicity islands which if activated could adulterate the recombinant product with dangerous substances. Transposible elements and other forms of selfish DNA that render the E. clol genome unstable could also cause difficulties with quality control. Some genes whose expression is simply unnecessary are therefore potentially wasteful. To improve the situation we have deleted over 14% of the E. coli K-12 genome in 39 segments. The reduced genome strain grows at a normal rate in minimal medium. By design it lacks all transposible elements, most candidates for toxic products and many seemingly unnecessary genes. A further reduction of the genome to 19.4% is in progress. In this research we propose to study the properties of the reduced genome E. coli relevant to the use as a production host by comparing the yield, purity and stability of a test product, Green Fluorescent Protein with the undeleted control strains DH10B and the parent MG1655. We also plan to measure the yield of DNA, and mutation frequency, and gene expression levels through DNA microarrays.
