Development of the selection tools for the evolutionary engineering of genetic networks and for parallel and continuous editing of genomic DNA

Directed evolution is a powerful strategy to construct complex yet functional systems ranging from genetic switch, regulatory circuits, and metabolic networks. To facilitate and accelerate the directed evolution of genetic switches and their assembly, we developed and tested various types of positive/negative selection systems for improving switching stringency, PoPS values, and rapidity in on/off transition. These methods were also adapted to the genome engineering, enabling the parallel, continuous, and seamless rewriting of multiple locus of bacterial genome.

1. Evolving for stringent switches and circuits
Reliable genetic networks require stringency in switch components. We developed a novel selection system using a viral nucleoside kinase as a single-gene dual selector. A single round of ON/OFF selection, all conducted by liquid handling, allowed us to enrich genetic circuitry with desired specification from variant pools, by the factor of 30,000x. Due to this power of the OFF selection, we could have isolated stringent circuits from the mixture containing non-stringent (leaky) ones.

2. Evolving temporal behaviours of genetic circuits
Synthetic biologists have been creating various timing circuits such as oscillators, pulse-generators, and delay switches (timers). For enabling the evolutionary design of temporal behaviour of the genetic circuits, we have developed another system, where both ON and OFF selections complete within 15 minutes. Using this selection, we have successfully enriched the rapid switchers, pulsegenerating circuits with desired time-profiles, and so on from the variant pools of the slowswitchers. Due to the rapidity of the selection process, one can also conduct serial operation of ON/OFF selections with reasonable time resolution to evolve temporal behaviour of the genetic circuits.

3. A method for continuous, iterative, and parallel operation of genome engineering
For the direct editing of bacterial genome with single-base resolution, two-step recombination methods are widely used. Here, the target site is replaced with PCR-generated DNA cassette first, and the cassette is then replaced with exogenous genes. Here, I report the systematic effort to redesign the system to improve the robustness, throughput, and speed of this method to realize the seamless and automation-compliant platform for genome engineering. Creating/testing the chimeric selectors, duplication of selector genes, tuning of the expression level of individual selector genes enabled us to eliminate the emergence of false-positive clones during the process. Having established the repeatable workflow where all the steps can be rapidly and seamlessly operated by liquid handling, we demonstrate the multi-step and in-parallel operation of genome modifications to generates a diverse set of isoprenoid-overproducing Escherichia coli strains.