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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

Loss-of-function mutations and evolutionary stability dynamics in T9002. (A) The T9002 genetic circuit. Symbols depict promoters (bent arrows), ribosome binding sites (ovals), coding sequences (arrows), and transcriptional terminators (octagons). T9002 consists of two devices, a luxR receiver device and a GFP-expressing device. The first device is composed of the tetR-regulated promoter R0040 that is constitutively expressed in the MG1655 strain since it does not produce TetR, B0034 RBS, C0062 luxR coding sequence, and B0010-B0012 (B0015) transcriptional terminator. The second device is composed of the R0062 luxR promoter, B0032 RBS, E0040 GFP coding sequence, and B0015 transcriptional terminator. LuxR is constitutively expressed from the tetR promoter. When the inducer 3OC6HSL (AHL) is added to the media, it binds with LuxR to activate transcription of GFP from the luxR promoter. If no AHL is in the media, the circuit is off. (B) Evolutionary stability dynamics of T9002 evolved under low (-AHL) and high (+AHL) input conditions. Low and high input evolved populations are shown with solid gray triangles and solid black circles, respectively. Evolved populations at different timepoints were grown with AHL to measure relative GFP levels. Relative fluorescence normalized by OD is plotted vs. generations. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) This circuit repeatedly has a deletion between homologous repeated terminators after 30 generations in the high input evolved populations.
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Figure 1: Loss-of-function mutations and evolutionary stability dynamics in T9002. (A) The T9002 genetic circuit. Symbols depict promoters (bent arrows), ribosome binding sites (ovals), coding sequences (arrows), and transcriptional terminators (octagons). T9002 consists of two devices, a luxR receiver device and a GFP-expressing device. The first device is composed of the tetR-regulated promoter R0040 that is constitutively expressed in the MG1655 strain since it does not produce TetR, B0034 RBS, C0062 luxR coding sequence, and B0010-B0012 (B0015) transcriptional terminator. The second device is composed of the R0062 luxR promoter, B0032 RBS, E0040 GFP coding sequence, and B0015 transcriptional terminator. LuxR is constitutively expressed from the tetR promoter. When the inducer 3OC6HSL (AHL) is added to the media, it binds with LuxR to activate transcription of GFP from the luxR promoter. If no AHL is in the media, the circuit is off. (B) Evolutionary stability dynamics of T9002 evolved under low (-AHL) and high (+AHL) input conditions. Low and high input evolved populations are shown with solid gray triangles and solid black circles, respectively. Evolved populations at different timepoints were grown with AHL to measure relative GFP levels. Relative fluorescence normalized by OD is plotted vs. generations. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) This circuit repeatedly has a deletion between homologous repeated terminators after 30 generations in the high input evolved populations.

Mentions: The two circuits we used to measure the evolutionary stability dynamics and determine the loss-of-function mutations were T9002 (Figure 1a) and I7101 (Figure 2a). T9002 is the Lux receiver circuit previously described [17] and expresses luxR that activates GFP expression when the inducer AHL is added to the media (see Figure 1 legend for details). I7101 has a lacI-regulated promoter and expresses GFP only when the inducer IPTG is added to the media since lacI is constitutively overexpressed from the chromosome in this particular strain (Escherichia coli MG1655 Z1). The evolutionary stability dynamics were measured by serial propagation with a dilution factor that allows for about 10 generations per day.


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 T9002. (A) The T9002 genetic circuit. Symbols depict promoters (bent arrows), ribosome binding sites (ovals), coding sequences (arrows), and transcriptional terminators (octagons). T9002 consists of two devices, a luxR receiver device and a GFP-expressing device. The first device is composed of the tetR-regulated promoter R0040 that is constitutively expressed in the MG1655 strain since it does not produce TetR, B0034 RBS, C0062 luxR coding sequence, and B0010-B0012 (B0015) transcriptional terminator. The second device is composed of the R0062 luxR promoter, B0032 RBS, E0040 GFP coding sequence, and B0015 transcriptional terminator. LuxR is constitutively expressed from the tetR promoter. When the inducer 3OC6HSL (AHL) is added to the media, it binds with LuxR to activate transcription of GFP from the luxR promoter. If no AHL is in the media, the circuit is off. (B) Evolutionary stability dynamics of T9002 evolved under low (-AHL) and high (+AHL) input conditions. Low and high input evolved populations are shown with solid gray triangles and solid black circles, respectively. Evolved populations at different timepoints were grown with AHL to measure relative GFP levels. Relative fluorescence normalized by OD is plotted vs. generations. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) This circuit repeatedly has a deletion between homologous repeated terminators after 30 generations in the high input evolved populations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Loss-of-function mutations and evolutionary stability dynamics in T9002. (A) The T9002 genetic circuit. Symbols depict promoters (bent arrows), ribosome binding sites (ovals), coding sequences (arrows), and transcriptional terminators (octagons). T9002 consists of two devices, a luxR receiver device and a GFP-expressing device. The first device is composed of the tetR-regulated promoter R0040 that is constitutively expressed in the MG1655 strain since it does not produce TetR, B0034 RBS, C0062 luxR coding sequence, and B0010-B0012 (B0015) transcriptional terminator. The second device is composed of the R0062 luxR promoter, B0032 RBS, E0040 GFP coding sequence, and B0015 transcriptional terminator. LuxR is constitutively expressed from the tetR promoter. When the inducer 3OC6HSL (AHL) is added to the media, it binds with LuxR to activate transcription of GFP from the luxR promoter. If no AHL is in the media, the circuit is off. (B) Evolutionary stability dynamics of T9002 evolved under low (-AHL) and high (+AHL) input conditions. Low and high input evolved populations are shown with solid gray triangles and solid black circles, respectively. Evolved populations at different timepoints were grown with AHL to measure relative GFP levels. Relative fluorescence normalized by OD is plotted vs. generations. Error bars represent one standard deviation from the mean of nine independently evolved populations. (C) This circuit repeatedly has a deletion between homologous repeated terminators after 30 generations in the high input evolved populations.
Mentions: The two circuits we used to measure the evolutionary stability dynamics and determine the loss-of-function mutations were T9002 (Figure 1a) and I7101 (Figure 2a). T9002 is the Lux receiver circuit previously described [17] and expresses luxR that activates GFP expression when the inducer AHL is added to the media (see Figure 1 legend for details). I7101 has a lacI-regulated promoter and expresses GFP only when the inducer IPTG is added to the media since lacI is constitutively overexpressed from the chromosome in this particular strain (Escherichia coli MG1655 Z1). The evolutionary stability dynamics were measured by serial propagation with a dilution factor that allows for about 10 generations per day.

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