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Designing and engineering evolutionary robust genetic circuits.

Sleight SC, Bartley BA, Lieviant JA, Sauro HM - J Biol Eng (2010)

Bottom Line: 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.We also found that on average, evolutionary half-life exponentially decreases with increasing expression levels.Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. sleight@u.washington.edu.

ABSTRACT

Background: One 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.

Results: We 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.

Conclusions: A 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.

No MeSH data available.


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Loss-of-function mutations and evolutionary stability dynamics in re-engineered T9002 circuits. (A) T9002 re-engineering involves changing the second double transcriptional terminator with varying degrees of homology and orientation to the first double transcriptional terminator. (B) Evolutionary stability dynamics of T9002 (solid black circles) and T9002 re-engineered circuits (various shapes and colors) under high input (+AHL) conditions. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) Types of mutations in nine independently evolved populations. For nine independently evolved populations, colored boxes correspond to the mutation legend below the table. The most common mutation for a particular type of mutation is labeled with "1" in the boxes above and less common mutations are labeled with increasing numbers. (D) Most common loss-of-function mutations that inactivated the re-engineered T9002 circuits. See Additional File 1, Supplementary Table S1 for mutation details.
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Figure 3: Loss-of-function mutations and evolutionary stability dynamics in re-engineered T9002 circuits. (A) T9002 re-engineering involves changing the second double transcriptional terminator with varying degrees of homology and orientation to the first double transcriptional terminator. (B) Evolutionary stability dynamics of T9002 (solid black circles) and T9002 re-engineered circuits (various shapes and colors) under high input (+AHL) conditions. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) Types of mutations in nine independently evolved populations. For nine independently evolved populations, colored boxes correspond to the mutation legend below the table. The most common mutation for a particular type of mutation is labeled with "1" in the boxes above and less common mutations are labeled with increasing numbers. (D) Most common loss-of-function mutations that inactivated the re-engineered T9002 circuits. See Additional File 1, Supplementary Table S1 for mutation details.

Mentions: Figure 1b shows the evolutionary stability dynamics of the T9002 circuit propagated in high input (with AHL) and low input (without AHL) conditions. From different timepoints in the experiment, the low and high input populations were induced with AHL to measure their normalized expression (here measured by fluorescence divided by cell density) over time. The low input evolved populations slowly lose their function to about 50% of the maximum after 300 generations. The evolved populations in high input conditions rapidly lose their function in less than 30 generations (the dynamics of this evolutionary stability are described below in Figure 3). No functional clones were observed after 30 generations as determined by measurement of individual colonies. The mutation that repeatedly causes loss-of-function in the high input evolved populations is a deletion between two homologous transcriptional terminators (Figure 1c), the same mutation described in [17]. This mutation evidently occurs at such a high rate that mutants quickly take over the population. In fact, Canton et al (2008) [17] were unable to isolate a population derived from a single isolate that did not already carry the deletion. The mutant plasmid was transformed back into the progenitor and was shown not to fluoresce after induction with AHL. In this initial study we also tested the evolutionary stability of a BioBrick engineered version of the repressilator circuit [1]. We could not measure its function over time due to unstable GFP expression at the population level, but found that the circuit repeatedly had a deletion between homologous tetR promoters.


Designing and engineering evolutionary robust genetic circuits.

Sleight SC, Bartley BA, Lieviant JA, Sauro HM - J Biol Eng (2010)

Loss-of-function mutations and evolutionary stability dynamics in re-engineered T9002 circuits. (A) T9002 re-engineering involves changing the second double transcriptional terminator with varying degrees of homology and orientation to the first double transcriptional terminator. (B) Evolutionary stability dynamics of T9002 (solid black circles) and T9002 re-engineered circuits (various shapes and colors) under high input (+AHL) conditions. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) Types of mutations in nine independently evolved populations. For nine independently evolved populations, colored boxes correspond to the mutation legend below the table. The most common mutation for a particular type of mutation is labeled with "1" in the boxes above and less common mutations are labeled with increasing numbers. (D) Most common loss-of-function mutations that inactivated the re-engineered T9002 circuits. See Additional File 1, Supplementary Table S1 for mutation details.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2991278&req=5

Figure 3: Loss-of-function mutations and evolutionary stability dynamics in re-engineered T9002 circuits. (A) T9002 re-engineering involves changing the second double transcriptional terminator with varying degrees of homology and orientation to the first double transcriptional terminator. (B) Evolutionary stability dynamics of T9002 (solid black circles) and T9002 re-engineered circuits (various shapes and colors) under high input (+AHL) conditions. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) Types of mutations in nine independently evolved populations. For nine independently evolved populations, colored boxes correspond to the mutation legend below the table. The most common mutation for a particular type of mutation is labeled with "1" in the boxes above and less common mutations are labeled with increasing numbers. (D) Most common loss-of-function mutations that inactivated the re-engineered T9002 circuits. See Additional File 1, Supplementary Table S1 for mutation details.
Mentions: Figure 1b shows the evolutionary stability dynamics of the T9002 circuit propagated in high input (with AHL) and low input (without AHL) conditions. From different timepoints in the experiment, the low and high input populations were induced with AHL to measure their normalized expression (here measured by fluorescence divided by cell density) over time. The low input evolved populations slowly lose their function to about 50% of the maximum after 300 generations. The evolved populations in high input conditions rapidly lose their function in less than 30 generations (the dynamics of this evolutionary stability are described below in Figure 3). No functional clones were observed after 30 generations as determined by measurement of individual colonies. The mutation that repeatedly causes loss-of-function in the high input evolved populations is a deletion between two homologous transcriptional terminators (Figure 1c), the same mutation described in [17]. This mutation evidently occurs at such a high rate that mutants quickly take over the population. In fact, Canton et al (2008) [17] were unable to isolate a population derived from a single isolate that did not already carry the deletion. The mutant plasmid was transformed back into the progenitor and was shown not to fluoresce after induction with AHL. In this initial study we also tested the evolutionary stability of a BioBrick engineered version of the repressilator circuit [1]. We could not measure its function over time due to unstable GFP expression at the population level, but found that the circuit repeatedly had a deletion between homologous tetR promoters.

Bottom Line: 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.We also found that on average, evolutionary half-life exponentially decreases with increasing expression levels.Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Bioengineering, University of Washington, Seattle, WA 98195, USA. sleight@u.washington.edu.

ABSTRACT

Background: One 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.

Results: We 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.

Conclusions: A 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.

No MeSH data available.


Related in: MedlinePlus