Plant & Animal Genomes XIV Conference

January 14-18, 2006
Town & Country Convention Center
San Diego, CA

A Reduced Genome Escherichia coli Lacking Mobile Elements Improves Cloning

David Frisch^1,2 , Gyorgy Posfai³ , Guy Plunkett III^1,2 , Tamas Feher³ , Guenther Keil⁴ , Monika de Arruda¹ , Kinga Umenhoffer³ , Vitaliy Kolisnychenko³ , Shamik Sharma⁵ , Sarah Harcum⁵ , Buffy Stahl¹ , John Campbell¹ , Frederick Blattner^1,2

¹  Scarab Genomics, Madison, WI, USA

²  Department of Genetics, University of Wisconsin, Madison, WI, USA

³  Institute of Biochemistry, Biological Research Center, Szeged, Hungary

⁴  Federal Research Centre for Virus Diseases of Animals, Institute of Molecular Biology, Friedrich-Loeffler Institutes, Greifswald-Insel Riems, Germany

⁵  Department of Bioengineering, Clemson University, Clemson, SC, USA

Scarless genomic deletions were used to delete 43 genomic regions from the sequenced E. coli strain, MG1655. Regions deleted include large K-islands, prophages, phage remnants, restriction modification genes, flagellar and chemotaxis related genes and all mobile elements including IS sequences and RHS (recombination hot spot) elements. Additional deletions include, EndA, recA, lac operon and tonA for T1 phage resistance. The resulting multiple deletion strain (MDS) has a 15.27% reduction in its genome from 4,639,221 to 3,930,956 bp. MDS strains were characterized for growth on standard microbiological media, transformation efficiency, protein expression, and mutation rates. The growth rate for the MDS strain is essentially unchanged relative to the parental strain MG1655 and the electroporation efficiency for pUC and BAC sized plasmids is equal to the best commercially available cells. Recombinant protein expression was observed to be similar for both MDS and MG1655 strains when chloramphenicol acetyl transferase (CAT) was expressed in cells grown to high densities. We found the frequency of IS insertion events increased 2.5-fold when CAT was over expressed in MG1655 whereas the mutation frequency caused by IS elements dropped to zero for the MDS strains. The observation that over expression of a protein induces IS transposition demonstrates that the MDS strain is a more stable host for protein production or library construction in which foreign proteins may be expressed. These improved traits make the MDS strain an ideal platform for large scale protein expression or library construction of both small and large inserts.