Rational optimization of tolC as a powerful dual selectable marker for genome engineering

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Counter-selection is crucial for genetic manipulation but historically lacks robustness and convenience.
• The TolC outer membrane pore in E. coli serves as a selectable/counter-selectable marker, but its use is limited by high counter-selection escape frequency with colicin E1.

Purpose of the Study:

• To identify mechanisms of counter-selection escape and engineer strains to reduce this frequency.
• To fundamentally improve the TolC counter-selection system for enhanced genetic manipulation in E. coli.
• To develop an optimized E. coli strain for efficient genome engineering and allelic diversity.

Main Methods:

• Unbiased deep sequencing of independent lineages to identify loss-of-function mutations conferring counter-selection escape.
• Strain engineering based on identified mutations to reduce counter-selection escape frequency.
• Supplementation of colicin E1 with vancomycin to enhance TolC counter-selection efficacy.
• Performance of stable, continuous selection/counter-selection rounds for multiple allele replacements.

Main Results:

• Strain engineering reduced TolC counter-selection escape frequency by approximately 40-fold.
• Supplementing with vancomycin reduced counter-selection escape by 425-fold compared to colicin E1 alone.
• Combined improvements in a mismatch repair proficient strain reduced counter-selection escape by 1.3E6-fold.
• Enabled replacement of 10 alleles without genotypic screening, demonstrating robust selection/counter-selection.
• Developed an optimized E. coli strain achieving approximately 10-fold greater allelic diversity.

Conclusions:

• The improved TolC counter-selection system is now as effective as most selectable markers, significantly enhancing the genome editing toolbox.
• This advancement facilitates stable and continuous genetic manipulation, enabling complex allele replacements efficiently.
• The optimized E. coli strain represents a significant improvement for high-throughput genome engineering and achieving allelic diversity.