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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.


Related in: MedlinePlus

Evolutionary half-life vs. initial expression level in T9002, T9002-E, R0011 + E0240, and R0010 + E0240 circuits evolved with different inducer concentrations. (A) Evolutionary half-life vs. initial expression level is plotted in T9002 (solid black circles) and T9002-E (solid dark red diamonds) circuits evolved with different AHL concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations. (B) Evolutionary half-life vs. initial expression level is plotted in R0011 + E0240 (solid black circles) and R0010 + E0240 (solid red circles) circuits evolved with different IPTG concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations.
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Figure 7: Evolutionary half-life vs. initial expression level in T9002, T9002-E, R0011 + E0240, and R0010 + E0240 circuits evolved with different inducer concentrations. (A) Evolutionary half-life vs. initial expression level is plotted in T9002 (solid black circles) and T9002-E (solid dark red diamonds) circuits evolved with different AHL concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations. (B) Evolutionary half-life vs. initial expression level is plotted in R0011 + E0240 (solid black circles) and R0010 + E0240 (solid red circles) circuits evolved with different IPTG concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations.

Mentions: The results of these experiments are shown in Figure 7. Figure 7a shows the mean initial expression level vs. mean evolutionary half-life for eight replicate populations from three different AHL concentrations in T9002 (black) and T9002-E (red). An exponential fit of these mean data points gives a much higher r2 value than a linear fit (> 0.1) in both cases. T9002 has an r2 value of 0.954 compared to the r2 value of 0.955 in T9002-E. The curve for T9002 is shifted to the left from T9002-E due to its higher mutation rate (expression alone cannot account for the shift), but as expression is decreased the evolutionary half-life difference between these two circuits also decreases. This decrease may be because at high expression levels, the fitness difference between the progenitor and mutant cells is the highest, and therefore mutants outcompete functional cells in the population more quickly.


Designing and engineering evolutionary robust genetic circuits.

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

Evolutionary half-life vs. initial expression level in T9002, T9002-E, R0011 + E0240, and R0010 + E0240 circuits evolved with different inducer concentrations. (A) Evolutionary half-life vs. initial expression level is plotted in T9002 (solid black circles) and T9002-E (solid dark red diamonds) circuits evolved with different AHL concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations. (B) Evolutionary half-life vs. initial expression level is plotted in R0011 + E0240 (solid black circles) and R0010 + E0240 (solid red circles) circuits evolved with different IPTG concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Evolutionary half-life vs. initial expression level in T9002, T9002-E, R0011 + E0240, and R0010 + E0240 circuits evolved with different inducer concentrations. (A) Evolutionary half-life vs. initial expression level is plotted in T9002 (solid black circles) and T9002-E (solid dark red diamonds) circuits evolved with different AHL concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations. (B) Evolutionary half-life vs. initial expression level is plotted in R0011 + E0240 (solid black circles) and R0010 + E0240 (solid red circles) circuits evolved with different IPTG concentrations. An exponential fit for the mean of each evolutionary half-life vs. initial expression data point is shown by the black line. Error bars for both the x and y-axis represent one standard deviation from the mean of eight independently evolved populations.
Mentions: The results of these experiments are shown in Figure 7. Figure 7a shows the mean initial expression level vs. mean evolutionary half-life for eight replicate populations from three different AHL concentrations in T9002 (black) and T9002-E (red). An exponential fit of these mean data points gives a much higher r2 value than a linear fit (> 0.1) in both cases. T9002 has an r2 value of 0.954 compared to the r2 value of 0.955 in T9002-E. The curve for T9002 is shifted to the left from T9002-E due to its higher mutation rate (expression alone cannot account for the shift), but as expression is decreased the evolutionary half-life difference between these two circuits also decreases. This decrease may be because at high expression levels, the fitness difference between the progenitor and mutant cells is the highest, and therefore mutants outcompete functional cells in the population more quickly.

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