Prolonging genetic circuit stability through adaptive evolution of overlapping genes

Abstract

The development of synthetic biological circuits that maintain functionality over application-relevant time scales remains a significant challenge. Here, we employed synthetic overlapping sequences in which one gene is encoded or 'entangled' entirely within an alternative reading frame of another gene. In this design, the toxin-encoding relE was entangled within ilvA, which encodes threonine deaminase, an enzyme essential for isoleucine biosynthesis. A functional entanglement construct was obtained upon modification of the ribosome-binding site of the internal relE gene. Using this optimized design, we found that the selection pressure to maintain functional IlvA stabilized the production of burdensome RelE for >130 generations, which compares favorably with the most stable kill-switch circuits developed to date. This stabilizing effect was achieved through a complete alteration of the allowable landscape of mutations such that mutations inactivating the entangled genes were disfavored. Instead, the majority of lineages accumulated mutations within the regulatory region of ilvA. By reducing baseline relE expression, these more 'benign' mutations lowered circuit burden, which suppressed the accumulation of relE-inactivating mutations, thereby prolonging kill-switch function. Overall, this work demonstrates the utility of sequence entanglement paired with an adaptive laboratory evolution campaign to increase the evolutionary stability of burdensome synthetic circuits.

Graphical Abstract

Figure 1.

Structure and genetic sequence components of the ilvA/relE entanglement. (A) Structure of WT E. coli threonine deaminase (blue) with the location of the relE sequence within the +1 frame of the ilvA/relE entanglement highlighted in red. The protein domains of IlvA are labeled and the region of amino acid changes imparted by the RBS modifications in ilvA/relE^RBSX constructs are shown in orange. The Protein Data Bank (PDB) accession number is 1TDJ. Structural renderings were generated using PyMOL v. 2.3.4. (B) Diagram of genetic circuit components used in this study. P. protegens Pf-5 was made auxotrophic for isoleucine via ΔilvA and ΔtdcB chromosomal deletions (right). Addition of cumate induces antitoxin expression by relieving CymR repression of chromosomal P_cymR-relB (right), while addition of rhamnose increases expression of ilvA/relE (or related alleles) through activation of plasmid-borne RhaR and RhaS, which activate P_rhaBAD (left).

Figure 2.

Internal RBS modifications improve functionality of ilvA/relE. (A) To probe ilvA function, strains harboring ilvA/relE^STOP alleles containing different strength RBSs were grown in minimal medium without isoleucine and without addition of rhamnose. In the diagram, stop codons in relE are represented by asterisks, and dashed lines indicate a non-functional relE. (B) Strains harboring ilvA/relE alleles with different strength internal RBSs were grown in rich medium to assess relE activity. To rescue growth, the antitoxin was induced by addition of cumate. For (A) and (B), strains harboring ilvA/relE vectors are listed in order of increasing internal RBS strength (see Supplementary Figure S5 for more details on RBS modifications). Growth is reported as OD₆₀₀ after 15 h. Data are shown as the mean ± standard deviation (SD) of three independent replicates.

Figure 3.

Escape ratio and the allowable landscape of mutations for ilvA/relE^RBS3 following a shift from permissive to non-permissive conditions. (A) Diagram of the procedure used to determine the escape ratio. Single isolates of the strain ΔilvAΔtdcB P_cymR-relB harboring a vector with ilvA/relE^RBS3 were grown under permissive conditions (LB + cumate) and then plated for CFU on minimal medium under both non-permissive (toxin-induced) and permissive (anti-toxin-induced) conditions with isoleucine. For each replicate, 48 colonies were then patched from the non-permissive plate with isoleucine onto non-permissive plates either with or without isoleucine. (B) The escape ratio was used as a metric of relE function and was determined by dividing the CFU/ml on non-permissive medium by the CFU/ml on permissive medium with isoleucine before patching. This was then multiplied by the proportion of colonies that survived on the patched plates either with isoleucine (+ile) or without isoleucine (–ile), respectively. An example of this proportion is seen in (A). Data are shown as the mean ± SD of eight independent replicates. Comparisons were made by Student's t-test. ***P < 0.001. (C) Schematic showing the types of mutations present in toxin escape colonies isolated from non-permissive patch plates without (top) or with isoleucine supplementation (bottom).

Figure 4.

The ilvA/relE^RBS3 entanglement increases the evolutionary stability of relE. (A) Independent lineages of ΔilvAΔtdcB P_cymR-relB harboring ilvA/relE^RBS3 on a vector were grown in minimal medium with rhamnose (to induce ilvA/relE^RBS3) and cumate (to induce relB antitoxin) in the presence (purple) or absence (green) of isoleucine. Each day (∼6.6 generations) the cultures were diluted 1:1000 in fresh medium and plated for CFU in toxin-permissive and non-permissive conditions, and an escape ratio was calculated. After 10 days, measurements were taken less frequently (every 5 days). Passaging in medium with isoleucine was discontinued at ∼66 generations when the population was stabilized with a near 100% escape ratio. Each condition is represented by 10 independent lineages which started from single colonies and are numbered in order of increasing final escape ratio. The gray bar indicates a toxin escape ratio ≥ 10⁻¹ (10%). (B) Average number of generations elapsed when the escape ratio exceeded 10⁻¹ (i.e. 10%, gray bar in A). Raw data for relE^RBS3 can be found in Supplementary Figure S9. The result for ilvA/relE^RBS3 grown without isoleucine is an underestimate as three lineages from this condition never reached 10% escape and were excluded from the calculation. Bar graph data are shown as the mean ± SD. Asterisks directly above bars denote comparisons with the ilvA/relE^RBS3 without isoleucine condition (– ile). Comparisons were made by one-way ANOVA with Tukey’s post-hoc test. ***P < 0.001.

Figure 5.

The ilvA/relE^RBS3 entanglement alters the allowable landscape of mutations and maintains the function of both genes. (A) Schematic of the types of mutations present in colonies isolated from each lineage of the long-term evolutionary stability assay grown either with or without isoleucine. A single colony was isolated from each lineage under permissive conditions after the final passage. Mutations were identified by sequencing the entire vector and the P_cymR-relB chromosomal region. Sequencing results from each isolate are listed in order of increasing final escape ratio seen in Figure 4A (isolates 1–10). The isolated vectors were transformed into a clean genetic background (ΔilvAΔtdcB P_cymR-relB) and were grown in (B) minimal medium without isoleucine and with addition of rhamnose and cumate to probe ilvA function or in (C) minimal medium with isoleucine and rhamnose and without cumate to test relE toxicity. For comparison with these clean background (CB) strains, the original parent strain (ilvA/relE^RBS3) and a strain in which the regulators rhaR and rhaS are deleted from the vector (ΔrhaR/rhaS) are included. Growth is reported as OD₆₀₀ after 15 h. Data are shown as the mean ± SD of three independent replicates.

Figure 6.

Sequence entanglement allows for circuit optimization through adaptive evolution. (A) Competition assay in which the parent strain ilvA/relE^RBS3 was grown in a 1:1 co-culture with clean background strains harboring vectors isolated from the listed lineages (CB 2–4). Strains were differentially marked with chromosomally integrated antibiotic cassettes, tetracycline and gentamicin. The competitive index was calculated as the CFU ratio of mutant/parent after growth for 48 h divided by the CFU ratio of the mutant/parent in the initial inoculum. (B) Assay of promoter activity for vectors isolated from lineages 1–7 of the long-term evolutionary stability assay. The gfp gene was cloned downstream of the promoter of each vector, replacing ilvA/relE, and strains were grown in minimal medium overnight with rhamnose. RFU is reported relative to OD₆₀₀. (C) Independent lineages of CB 2—a clean genetic background (ΔilvAΔtdcB P_cymR-relB) strain harboring the ilvA/relE^RBS3 vector isolated from the original lineage 2 in Figure 4B—were grown in minimal medium with rhamnose (to induce ilvA/relE^RBS3) and cumate (to induce antitoxin) in the presence (purple) or absence (green) of isoleucine. Every 5 days (∼33 generations) the cultures were diluted 1:1000 in fresh medium and plated for CFU under toxin-permissive and non-permissive conditions, and an escape ratio was calculated. Each condition is represented by 10 independent lineages which started from single colonies grown on permissive medium. Data for (A) and (B) are shown as the mean ± SD of 3–4 independent replicates. Asterisk(s) directly above data denote comparisons with the parent condition. Comparisons were made by one-way ANOVA with Dunnet's (A) or Tukey's (B) post-hoc test. ***P < 0.001, **P < 0.01, *P < 0.05.