Ribozyme-based insulator parts buffer synthetic circuits from genetic context

Abstract

Synthetic genetic programs are built from circuits that integrate sensors and implement temporal control of gene expression1,2,3,4. Transcriptional circuits are layered by using promoters to carry the signal between circuits. In other words, the output promoter of one circuit serves as the input promoter to the next. Thus, connecting circuits requires physically connecting a promoter to the next circuit. We show that the sequence at the junction between the input promoter and circuit can affect the input-output response (transfer function) of the circuit5,6,7,8,9. A library of putative sequences that might reduce (or buffer) such context effects, which we refer to as 'insulator parts', is screened in Escherichia coli. We find that ribozymes that cleave the 5′ untranslated region (5′-UTR) of the mRNA are effective insulators. They generate quantitatively identical transfer functions, irrespective of the identity of the input promoter. When these insulators are used to join synthetic gene circuits, the behavior of layered circuits can be predicted using a mathematical model. The inclusion of insulators will be critical in reliably permuting circuits to build different programs.

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Figure 1: The transfer function of the NOT gate depends on the inducible system used to measure it.
Figure 2: Screening the library of insulator parts.
Figure 3: RiboJ and other insulators insulate the transfer function of the NOT and BUFFER gates.

References

  1. 1

    Kim, P.M. & Tidor, B. Limitations of quantitative gene regulation models: a case study. Genome Res. 13, 2391–2395 (2003).

    CAS  Article  Google Scholar 

  2. 2

    Del Vecchio, D., Ninfa, A.J. & Sontag, E.D. Modular cell biology: retroactivity and insulation. Mol. Syst. Biol. 4, 161 (2008).

    Article  Google Scholar 

  3. 3

    Grünberg, R. & Serrano, L. Strategies for protein synthetic biology. Nucleic Acids Res. 38, 2663–2675 (2010).

    Article  Google Scholar 

  4. 4

    Tan, C., Marguet, P. & You, L. Emergent bistability by a growth-modulating positive feedback circuit. Nat. Chem. Biol. 5, 842–848 (2009).

    CAS  Article  Google Scholar 

  5. 5

    Ellis, T., Wang, X. & Collins, J.J. Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat. Biotechnol. 27, 465–471 (2009).

    CAS  Article  Google Scholar 

  6. 6

    Anderson, J.C., Voigt, C.A. & Arkin, A.P. Environmental signal integration by a modular AND gate. Mol. Syst. Biol. 3, 133 (2007).

    Article  Google Scholar 

  7. 7

    Wang, B., Kitney, R.I., Joly, N. & Buck, M. Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology. Nat. Commun. 2, 508 (2011).

    Article  Google Scholar 

  8. 8

    Hooshangi, S., Thiberge, S. & Weiss, R. Ultrasensitivity and noise propagation in a synthetic transcriptional cascade. Proc. Natl. Acad. Sci. USA 102, 3581–3586 (2005).

    CAS  Article  Google Scholar 

  9. 9

    Pedraza, J.M. & van Oudenaarden, A. Noise propagation in gene networks. Science 307, 1965–1969 (2005).

    CAS  Article  Google Scholar 

  10. 10

    Endy, D. Foundations for engineering biology. Nature 438, 449–453 (2005).

    CAS  Article  Google Scholar 

  11. 11

    Voigt, C.A. Genetic parts to program bacteria. Curr. Opin. Biotechnol. 17, 548–557 (2006).

    CAS  Article  Google Scholar 

  12. 12

    Bio FAB Group et al. Engineering life: building a fab for biology. Sci. Am. 294, 44–51 (2006).

  13. 13

    Shetty, R.P., Endy, D. & Knight, T.F. Jr Engineering BioBrick vectors from BioBrick parts. J. Biol. Eng. 2, 5 (2008).

    Article  Google Scholar 

  14. 14

    Anderson, J.C. et al. BglBricks: A flexible standard for biological part assembly. J. Biol. Eng. 4, 1 (2010).

    Article  Google Scholar 

  15. 15

    Clancy, K. & Voigt, C.A. Programming cells: towards an automated 'Genetic Compiler'. Curr. Opin. Biotechnol. 21, 572–581 (2010).

    CAS  Article  Google Scholar 

  16. 16

    Arkin, A. Setting the standard in synthetic biology. Nat. Biotechnol. 26, 771–774 (2008).

    CAS  Article  Google Scholar 

  17. 17

    Rhodius, V.A., Mutalik, V.K. & Gross, C.A. Predicting the strength of UP-elements and full-length E. coli sE promoters. Nucleic Acids Res. 10.1093/nar/gkr1190 (2012).

  18. 18

    Davis, J.H., Rubin, A.J. & Sauer, R.T. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 39, 1131–1141 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Carrier, T.A. & Keasling, J.D. Engineering mRNA stability in E. coli by the addition of synthetic hairpins using a 5′ cassette system. Biotechnol. Bioeng. 55, 577–580 (1997).

    CAS  Article  Google Scholar 

  20. 20

    Qi, L., Haurwitz, R.E., Shao, W., Doudna, J.A. & Arkin, A.P. RNA processing enables predictable programming of gene expression. Nat. Biotechnol.advance online publication, 10.1038/nbt.2355 (16 September 2012).

  21. 21

    Gardner, T.S., Cantor, C.R. & Collins, J.J. Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339–342 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Elowitz, M.B. & Leibler, S. A synthetic oscillatory network of transcriptional regulators. Nature 403, 335–338 (2000).

    CAS  Article  Google Scholar 

  23. 23

    Yokobayashi, Y., Weiss, R. & Arnold, F.H. Directed evolution of a genetic circuit. Proc. Natl. Acad. Sci. USA 99, 16587–16591 (2002).

    CAS  Article  Google Scholar 

  24. 24

    Lou, C. et al. Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch. Mol. Syst. Biol. 6, 350 (2010).

    Article  Google Scholar 

  25. 25

    Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).

    CAS  Article  Google Scholar 

  26. 26

    Buzayan, J.M., Gerlach, W.L. & Bruening, G. Satellite tobacco ringspot virus RNA: A subset of the RNA sequence is sufficient for autolytic processing. Proc. Natl. Acad. Sci. USA 83, 8859–8862 (1986).

    CAS  Article  Google Scholar 

  27. 27

    Galdzicki, M., Rodriguez, C., Chandran, D., Sauro, H.M. & Gennari, J.H. Standard Biological Parts Knowledgebase. PLoS ONE 6, e17005 (2011).

    CAS  Article  Google Scholar 

  28. 28

    Forster, A.C. & Symons, R.H. Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell 50, 9–16 (1987).

    CAS  Article  Google Scholar 

  29. 29

    Di Serio, F., Daròs, J.A., Ragozzino, A. & Flores, R. A 451-nucleotide circular RNA from cherry with hammerhead ribozymes in its strands of both polarities. J. Virol. 71, 6603–6610 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Kaper, J.M., Tousignant, M.E. & Steger, G. Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus. Biochem. Biophys. Res. Commun. 154, 318–325 (1988).

    CAS  Article  Google Scholar 

  31. 31

    Hernández, C. & Flores, R. Plus and minus RNAs of peach latent mosaic viroid self-cleave in vitro via hammerhead structures. Proc. Natl. Acad. Sci. USA 89, 3711–3715 (1992).

    Article  Google Scholar 

  32. 32

    Roossinck, M.J., Sleat, D. & Palukaitis, P. Satellite RNAs of plant viruses: structures and biological effects. Microbiol. Rev. 56, 265–279 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Beal, J., Lu, T. & Weiss, R. Automatic compilation from high-level biologically-oriented programming language to genetic regulatory networks. PLoS ONE 6, e22490 (2011).

    CAS  Article  Google Scholar 

  34. 34

    Munsky, B., Neuert, G. & van Oudenaarden, A. Using gene expression noise to understand gene regulation. Science 336, 183–187 (2012).

    CAS  Article  Google Scholar 

  35. 35

    Gillespie, D.T. Exact stochastic simulation of coupled chemical reactions. J. Phys. Chem. 81, 2340–2361 (1977).

    CAS  Article  Google Scholar 

  36. 36

    Arkin, A., Ross, J. & McAdams, H.H. Stochastic kinetic analysis of developmental pathway bifurcation in phage l-infected Escherichia coli cells. Genetics 149, 1633–1648 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Munsky, B. & Khammash, M. The finite state projection algorithm for the solution of the chemical master equation. J. Chem. Phys. 124, 044104 (2006).

    Article  Google Scholar 

  38. 38

    Munsky, B., Trinh, B. & Khammash, M. Listening to the noise: random fluctuations reveal gene network parameters. Mol. Syst. Biol. 5, 318 (2009).

    Article  Google Scholar 

  39. 39

    Moon, T.S., Lou, C., Tamsir, A., Stanton B.C. & Voigt, C.A. Genetic programs constructed from layered logic gates in single cells. Nature advance online publication, 10.1038/nature11516 (7 October 2012).

  40. 40

    Walczak, A.M., Mugler, A. & Wiggins, C.H. A stochastic spectral analysis of transcriptional regulatory cascades. Proc. Natl. Acad. Sci. USA 106, 6529–6534 (2009).

    CAS  Article  Google Scholar 

  41. 41

    Ro, D.-K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature 440, 940–943 (2006).

    CAS  Article  Google Scholar 

  42. 42

    Temme, K., Zhao, D. & Voigt, C.A. Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc. Natl. Acad. Sci. USA 109, 7085–7090 (2012).

    Article  Google Scholar 

  43. 43

    Rhodius, V.A. & Mutalik, V.K. Predicting strength and function for promoters of the Escherichia coli alternative sigma factor, sigmaE. Proc. Natl. Acad. Sci. USA 107, 2854–2859 (2010).

    CAS  Article  Google Scholar 

  44. 44

    Salis, H.M., Mirsky, E.A. & Voigt, C.A. Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946–950 (2009).

    CAS  Article  Google Scholar 

  45. 45

    Delcher, A.L., Harmon, D., Kasif, S., White, O. & Salzberg, S.L. Improved microbial gene identification with GLIMMER. Nucleic Acids Res. 27, 4636–4641 (1999).

    CAS  Article  Google Scholar 

  46. 46

    Kelly, J.R. et al. Measuring the activity of BioBrick promoters using an in vivo reference standard. J. Biol. Eng. 3, 4 (2009).

    Article  Google Scholar 

  47. 47

    Khvorova, A., Lescoute, A., Westhof, E. & Jayasena, S.D. Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nat. Struct. Biol. 10, 708–712 (2003).

    CAS  Article  Google Scholar 

  48. 48

    Nelson, J.A., Shepotinovskaya, I. & Uhlenbeck, O.C. Hammerheads derived from sTRSV show enhanced cleavage and ligation rate constants. Biochemistry 44, 14577–14585 (2005).

    CAS  Article  Google Scholar 

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Acknowledgements

C.A.V. is supported by Life Technologies, Defense Advanced Research Projects Agency Chronicle of Lineage Indicative of Origins (DARPA CLIO, N66001-12-C-4018), Office of Naval Research (N00014-10-1-0245), National Science Foundation (NSF) (CCF-0943385), National Institutes of Health (AI067699) and the NSF Synthetic Biology Engineering Research Center (SynBERC, SA5284-11210).

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C.A.V. conceived of and supervised the project. C.L. designed and performed the experiments. B.S. performed experiments with the McbR repressor. Y.-J.C. and C.L. performed the q-PCR experiments. B.M. and C.L. analyzed data. C.L., B.M., Y.-J.C. and C.A.V. wrote the manuscript.

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Correspondence to Christopher A Voigt.

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The authors declare no competing financial interests.

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Lou, C., Stanton, B., Chen, Y. et al. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nat Biotechnol 30, 1137–1142 (2012). https://doi.org/10.1038/nbt.2401

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