Designing and engineering evolutionary robust genetic circuits

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.

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.

Figure 2

Loss-of-function mutations and evolutionary stability dynamics in I7101. (A) The I7101 genetic circuit. Symbols depict promoters (bent arrows), ribosome binding sites (ovals), coding sequences (arrows), and transcriptional terminators (octagons). I7101 consists of the promoter R0011 and the GFP-expressing element E0240 that is made up of the B0032 RBS, E0040 GFP coding sequence, and B0010-B0012 (B0015) transcriptional terminator. Since lacI is constitutively expressed from the chromosome, it represses GFP expression from the lacI-regulated promoter R0011. When the inducer Isopropyl-beta-D-thiogalactopyranoside (IPTG) is added to the media, it inhibits LacI and activates GFP expression. If no IPTG is in the media, the circuit is off. (B) Evolutionary stability dynamics of R0011 + E0240 evolved under low (-IPTG) and high (+IPTG) 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 IPTG 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 operators within R0011 after 90 generations in the high input evolved populations.

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.

Figure 4

Loss-of-function mutations and evolutionary stability dynamics in re-engineered I7101 circuits (A) I7101 (R0011 + E0240) re-engineering involves swapping out the R0011 promoter. (B) Evolutionary stability dynamics of R0011 + E0240 (open black circles) and re-engineered circuits (various shapes and colors) under constitutive expression. 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. See Additional File 1, Supplementary Table S1 for mutation details.

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.

Figure 6

Loss-of-function mutations and evolutionary stability dynamics in two lacI-regulated circuits under constitutive vs. inducible expression. (A) Regulation of inducible R0011 + E0240 and R0010 + E0240 circuits. LacI represses transcription of GFP and IPTG de-represses the circuit to allow for GFP expression. (B) Top panel shows the evolutionary stability dynamics of constitutive R0011 + E0240 (open black circles) and inducible R0011 + E0240 (solid black circles). Bottom panel shows the evolutionary stability dynamics of constitutive R0010 + E0240 (open red circles) and inducible R0010 + E0240 (solid red circles). 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 R0011 + E0240 and R0010 + E0240 inducible and constitutive circuits. See Additional File 1, Supplementary Table S1 for mutation details.

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.