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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 I7101 circuits with a kanamycin resistance gene. (A) I7101 re-engineering with the addition of a kanamycin resistance (kanR) gene. First the R0010 promoter was added instead of R0011 (top). Then, this circuit was re-engineered to polycistronically transcribe gfp and kanR separately into separate GFP and KanR proteins (middle) and to express a GFP-KanR fusion protein (bottom). (B) Top panel shows the evolutionary stability dynamics of R0010 + E0240 kanR polycistronic circuits propagated with kanamycin (solid green circles) and without kanamycin (open green circles). Bottom panel shows the evolutionary stability dynamics of R0010 + E0240 kanR fusion circuits propagated with kanamycin (solid blue circles) and without kanamycin (open blue circles). R0010 + E0240 and R0011 + E0240 evolutionary stability dynamics are shown in Figure 4. 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 I7101 circuits with a kanamycin resistance gene. See Additional File 1, Supplementary Table S1 for mutation details.
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Figure 5: Loss-of-function mutations and evolutionary stability dynamics in re-engineered I7101 circuits with a kanamycin resistance gene. (A) I7101 re-engineering with the addition of a kanamycin resistance (kanR) gene. First the R0010 promoter was added instead of R0011 (top). Then, this circuit was re-engineered to polycistronically transcribe gfp and kanR separately into separate GFP and KanR proteins (middle) and to express a GFP-KanR fusion protein (bottom). (B) Top panel shows the evolutionary stability dynamics of R0010 + E0240 kanR polycistronic circuits propagated with kanamycin (solid green circles) and without kanamycin (open green circles). Bottom panel shows the evolutionary stability dynamics of R0010 + E0240 kanR fusion circuits propagated with kanamycin (solid blue circles) and without kanamycin (open blue circles). R0010 + E0240 and R0011 + E0240 evolutionary stability dynamics are shown in Figure 4. 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 I7101 circuits with a kanamycin resistance gene. See Additional File 1, Supplementary Table S1 for mutation details.

Mentions: We also re-engineered the I7101 circuit to have a kanamycin resistance gene (kanR) in order to put a selective pressure on the promoter (Figure 5a). Both a GFP-KanR fusion coding sequence and polycistronic transcribed coding sequence were engineered (Figure 5a). We also swapped out the R0011 promoter with the R0010 promoter since it was found to be more evolutionary robust than R0011 (Figure 5a). The other parts of this figure show the evolutionary stability dynamics for these re-engineered circuits (Figure 5b), mutations found in the nine evolved populations (Figure 5c), and most common mutation for each re-engineered circuit (Figure 5d). These circuits were constitutively expressed and the KanR circuits were propagated with and without kanamycin (kan) in the media. We predicted that the kan propagated circuits would be more evolutionary robust than those without kan in the media.


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 I7101 circuits with a kanamycin resistance gene. (A) I7101 re-engineering with the addition of a kanamycin resistance (kanR) gene. First the R0010 promoter was added instead of R0011 (top). Then, this circuit was re-engineered to polycistronically transcribe gfp and kanR separately into separate GFP and KanR proteins (middle) and to express a GFP-KanR fusion protein (bottom). (B) Top panel shows the evolutionary stability dynamics of R0010 + E0240 kanR polycistronic circuits propagated with kanamycin (solid green circles) and without kanamycin (open green circles). Bottom panel shows the evolutionary stability dynamics of R0010 + E0240 kanR fusion circuits propagated with kanamycin (solid blue circles) and without kanamycin (open blue circles). R0010 + E0240 and R0011 + E0240 evolutionary stability dynamics are shown in Figure 4. 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 I7101 circuits with a kanamycin resistance gene. See Additional File 1, Supplementary Table S1 for mutation details.
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Figure 5: Loss-of-function mutations and evolutionary stability dynamics in re-engineered I7101 circuits with a kanamycin resistance gene. (A) I7101 re-engineering with the addition of a kanamycin resistance (kanR) gene. First the R0010 promoter was added instead of R0011 (top). Then, this circuit was re-engineered to polycistronically transcribe gfp and kanR separately into separate GFP and KanR proteins (middle) and to express a GFP-KanR fusion protein (bottom). (B) Top panel shows the evolutionary stability dynamics of R0010 + E0240 kanR polycistronic circuits propagated with kanamycin (solid green circles) and without kanamycin (open green circles). Bottom panel shows the evolutionary stability dynamics of R0010 + E0240 kanR fusion circuits propagated with kanamycin (solid blue circles) and without kanamycin (open blue circles). R0010 + E0240 and R0011 + E0240 evolutionary stability dynamics are shown in Figure 4. 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 I7101 circuits with a kanamycin resistance gene. See Additional File 1, Supplementary Table S1 for mutation details.
Mentions: We also re-engineered the I7101 circuit to have a kanamycin resistance gene (kanR) in order to put a selective pressure on the promoter (Figure 5a). Both a GFP-KanR fusion coding sequence and polycistronic transcribed coding sequence were engineered (Figure 5a). We also swapped out the R0011 promoter with the R0010 promoter since it was found to be more evolutionary robust than R0011 (Figure 5a). The other parts of this figure show the evolutionary stability dynamics for these re-engineered circuits (Figure 5b), mutations found in the nine evolved populations (Figure 5c), and most common mutation for each re-engineered circuit (Figure 5d). These circuits were constitutively expressed and the KanR circuits were propagated with and without kanamycin (kan) in the media. We predicted that the kan propagated circuits would be more evolutionary robust than those without kan in the media.

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