Abstract
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.
Highlights
Selectable markers have long been critical tools in molecular genetics, enabling the genetic manipulation of model organisms
72% of the population is unmodified and only 8.3% of the population harbors more than a single edit, yielding a population average of 0.43 ± 0.06 edits/clone/cycle (Figure 1B), demonstrating that repeated cycling is crucial to attain complex genotypes with Multiplex Automatable Genome Engineering (MAGE)
We demonstrated the mechanistic independence of vancomycin escape from colicin E1 (colE1) escape by showing that vancomycin selection was still effective on the 96 tolC counter-selection escape clones used for whole-genome re-sequencing (Figure 4B)
Summary
Selectable markers have long been critical tools in molecular genetics, enabling the genetic manipulation of model organisms. Classical selectable markers are often antibiotic resistance genes, such as aminoglycoside phosphotransferase (kanamycin resistance), whose gene products are required for growth in media containing kanamycin. Selectable markers are used for plasmid maintenance, engineered conjugation and genome manipulations [1]. Counter-selectable markers such as sacB [2] or barnase [3] are useful tools for different applications, such as plasmid curing [4,5], scar-less gene deletion [2] or engineering double-crossovers [6]. Counter-selectable markers often require stringent growth conditions to achieve robust counter-selection performance, and there are few means to ensure proper function of counter-selectable markers in vivo, which limit their application. Dual selectable markers are advantageous for generating gene replacements, scar-less genome editing and selection-coupled biosensors [7]. Several dual selectable markers have been reported [rpsL [8,9], galK [10], thyA [11], hsvTK [12], tetA [13] and tolC [14]], but as with counter-selectable markers, dual-selectable markers suffer from high counter-selection escape and/or reliance on minimal media for robust counter-selection [10]
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