Rational optimization of tolC as a powerful dual selectable marker for genome engineering.
Selection has been invaluable for genetic manipulation, although counter-selection has historically exhibited limited robustness and convenience. TolC, an outer membrane pore involved in transmembrane transport in E. coli, has been implemented as a selectable/counter-selectable marker, but counter-selection escape frequency using colicin E1 precludes using tolC for inefficient genetic manipulations and/or with large libraries. Here, we leveraged unbiased deep sequencing of 96 independent lineages exhibiting counter-selection escape to identify loss-of-function mutations, which offered mechanistic insight and guided strain engineering to reduce counter-selection escape frequency by ∼40-fold. We fundamentally improved the tolC counter-selection by supplementing a second agent, vancomycin, which reduces counter-selection escape by 425-fold, compared colicin E1 alone. Combining these improvements in a mismatch repair proficient strain reduced counter-selection escape frequency by 1.3E6-fold in total, making tolC counter-selection as effective as most selectable markers, and adding a valuable tool to the genome editing toolbox. These improvements permitted us to perform stable and continuous rounds of selection/counter-selection using tolC, enabling replacement of 10 alleles without requiring genotypic screening for the first time. Finally, we combined these advances to create an optimized E. coli strain for genome engineering that is ∼10-fold more efficient at achieving allelic diversity than previous best practices.
Department of Genetics and Wyss Institute for Biologically Inspired Engineering, Harvard Medical School, Boston, MA 02115, USA, Program in Chemical Biology, Harvard University, Cambridge, MA 02138, USA, Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA and Molecular, Cellular, Developmental and Systems Biology Institute, Yale University, New Haven, CT 06516, USA.
Christopher J
CJ
Gregg
Marc J
MJ
Lajoie
Michael G
MG
Napolitano
Joshua A
JA
Mosberg
Daniel B
DB
Goodman
John
J
Aach
Farren J
FJ
Isaacs
George M
GM
Church
D000483:Alleles; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D015415:Biomarkers; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics*; D017353:Gene Deletion; D020440:Gene Duplication; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D059014:High-Throughput Nucleotide Sequencing; D026901:Membrane Transport Proteins / Q000235:genetics*; D010641:Phenotype
D001425:Bacterial Outer Membrane Proteins; D015415:Biomarkers; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli