Designing and engineering evolutionary robust genetic circuits

Abstract

BackgroundOne problem with engineered genetic circuits in synthetic microbes is their stability over evolutionary time in the absence of selective pressure. Since design of a selective environment for maintaining function of a circuit will be unique to every circuit, general design principles are needed for engineering evolutionary robust circuits that permit the long-term study or applied use of synthetic circuits.ResultsWe first measured the stability of two BioBrick-assembled genetic circuits propagated in Escherichia coli over multiple generations and the mutations that caused their loss-of-function. The first circuit, T9002, loses function in less than 20 generations and the mutation that repeatedly causes its loss-of-function is a deletion between two homologous transcriptional terminators. To measure the effect between transcriptional terminator homology levels and evolutionary stability, we re-engineered six versions of T9002 with a different transcriptional terminator at the end of the circuit. When there is no homology between terminators, the evolutionary half-life of this circuit is significantly improved over 2-fold and is independent of the expression level. Removing homology between terminators and decreasing expression level 4-fold increases the evolutionary half-life over 17-fold. The second circuit, I7101, loses function in less than 50 generations due to a deletion between repeated operator sequences in the promoter. This circuit was re-engineered with different promoters from a promoter library and using a kanamycin resistance gene (kanR) within the circuit to put a selective pressure on the promoter. The evolutionary stability dynamics and loss-of-function mutations in all these circuits are described. We also found that on average, evolutionary half-life exponentially decreases with increasing expression levels.ConclusionsA wide variety of loss-of-function mutations are observed in BioBrick-assembled genetic circuits including point mutations, small insertions and deletions, large deletions, and insertion sequence (IS) element insertions that often occur in the scar sequence between parts. Promoter mutations are selected for more than any other biological part. Genetic circuits can be re-engineered to be more evolutionary robust with a few simple design principles: high expression of genetic circuits comes with the cost of low evolutionary stability, avoid repeated sequences, and the use of inducible promoters increases stability. Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.

Computational design of genomic transcriptional networks with adaptation to varying environments

“…In addition, as our GTRN evolution only relocates genes, it is difficult to change the mode of regulation in a single evolution step. We should notice that regulatory circuits designed in synthetic biology often lack robustness to mutational drift (24). Sometimes this lack of evolutionary robustness may be desired for biosafety reasons.…”

Promoter and Terminator Discovery and Engineering

“…As an example, commonly used terminators such as the native bacteriophage T7 terminator exhibit low termination efficiencies, meaning that transcriptional flux continues through the expression cassette and affects the regulation of downstream genes and limits polymerase recycling [113][114][115]. Furthermore, the collection of terminators available to researchers has traditionally been much smaller in breadth than promoters [116], thus limiting large-scale pathways and circuits because of the fear of genetic instability via homologous recombination [117,118]. Terminators also serve as a control point to tune expression in eukaryotes via the stability of the 3 0 end of the mRNA transcript [119][120][121].…”

Efficient Search, Mapping, and Optimization of Multi-protein Genetic Systems in Diverse Bacteria

“…Many genetic parts with similar expression levels must be available, together with many genetic parts that span the entire expression level range. Genetic parts must not have long repetitive sequences to minimize rates of homologous recombination and undesired navigation of the sequence-expression-activity space (Lovett, 2004;Sleight et al, 2010). By combining sequence-dependent biophysical models with optimization, we can generate an unlimited number of non-repetitive genetic parts that span the entire expression range, and in diverse bacterial hosts.…”