Probing the limits of genetic recoding in essential genes.

Engineering radically altered genetic codes will allow for genomically recoded organisms that have expanded chemical capabilities and are isolated from nature. We have previously reassigned the translation function of the UAG stop codon; however, reassigning sense codons poses a greater challenge because such codons are more prevalent, and their usage regulates gene expression in ways that are difficult to predict. To assess the feasibility of radically altering the genetic code, we selected a panel of 42 highly expressed essential genes for modification. Across 80 Escherichia coli strains, we removed all instances of 13 rare codons from these genes and attempted to shuffle all remaining codons. Our results suggest that the genome-wide removal of 13 codons is feasible; however, several genome design constraints were apparent, underscoring the importance of a strategy that rapidly prototypes and tests many designs in small pieces.

Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering.

Disrupting the interaction between primase and helicase in Escherichia coli increases Okazaki fragment (OF) length due to less frequent primer synthesis. We exploited this feature to increase the amount of ssDNA at the lagging strand of the replication fork that is available for λ Red-mediated Multiplex Automatable Genome Engineering (MAGE). Supporting this concept, we demonstrate that MAGE enhancements correlate with OF length. Compared with a standard recombineering strain (EcNR2), the strain with the longest OFs displays on average 62% more alleles converted per clone, 239% more clones with 5 or more allele conversions and 38% fewer clones with 0 allele conversions in 1 cycle of co-selection MAGE (CoS-MAGE) with 10 synthetic oligonucleotides. Additionally, we demonstrate that both synthetic oligonucleotides and accessible ssDNA targets on the lagging strand of the replication fork are limiting factors for MAGE. Given this new insight, we generated a strain with reduced oligonucleotide degradation and increased genomic ssDNA availability, which displayed 111% more alleles converted per clone, 527% more clones with 5 or more allele conversions and 71% fewer clones with 0 allele conversions in 1 cycle of 10-plex CoS-MAGE. These improvements will facilitate ambitious genome engineering projects by minimizing dependence on time-consuming clonal isolation and screening.

Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate.

The phage lambda-derived Red recombination system is a powerful tool for making targeted genetic changes in Escherichia coli, providing a simple and versatile method for generating insertion, deletion, and point mutations on chromosomal, plasmid, or BAC targets. However, despite the common use of this system, the detailed mechanism by which lambda Red mediates double-stranded DNA recombination remains uncertain. Current mechanisms posit a recombination intermediate in which both 5' ends of double-stranded DNA are recessed by λ exonuclease, leaving behind 3' overhangs. Here, we propose an alternative in which lambda exonuclease entirely degrades one strand, while leaving the other strand intact as single-stranded DNA. This single-stranded intermediate then recombines via beta recombinase-catalyzed annealing at the replication fork. We support this by showing that single-stranded gene insertion cassettes are recombinogenic and that these cassettes preferentially target the lagging strand during DNA replication. Furthermore, a double-stranded DNA cassette containing multiple internal mismatches shows strand-specific mutations cosegregating roughly 80% of the time. These observations are more consistent with our model than with previously proposed models. Finally, by using phosphorothioate linkages to protect the lagging-targeting strand of a double-stranded DNA cassette, we illustrate how our new mechanistic knowledge can be used to enhance lambda Red recombination frequency. The mechanistic insights revealed by this work may facilitate further improvements to the versatility of lambda Red recombination.