Abstract
Synthetic biology promises to enhance our ability to control biological systems by creating a systematic approach for the construction of genetic circuits that reliably program cellular function. As part of this approach, efficient methods are needed for the tuning of genetic circuits so as to allow for optimization of a design despite varying cellular contexts and incomplete understanding of in vivo biological interactions. Here we outline an optimization method that we have used to improve the logical responses of a genetic AND logic gate derived from components of the LuxI–LuxR bacterial quorum-sensing system. Basing our approach on the idea of evolutionary design, we improved the properties of our genetic AND logic gate by using directed evolution and a two-step screening process to alter the activities of the LuxR transcriptional activator. Using this method, we were able to rapidly enhance the AND gate’s logical responses and have increased the specificities of these responses by ∼1.5-fold.
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References
Andrianantoandro, E., Basu, S., Karig, D. K., and Weiss, R. (2006) Synthetic biology: New engineering rules for an emerging discipline. Mol. Syst. Biol. 2, 1–14.
Marguet, P., Balagadde, F., Tan, C., and You, L. (2007) Biology by design: Reduction and synthesis of cellular components and behaviour. J. R. Soc. Interface 4, 607–623.
Haseltine, E. L., and Arnold, F. H. (2007) Synthetic gene circuits: Design with directed evolution. Annu. Rev. Biophys. Biomol. Struct. 36, 1–19.
Yokobayashi, Y., Weiss, R., and Arnold, F. H. (2002) Directed evolution of a genetic circuit. Proc. Natl. Acad. Sci. USA 99, 16587–16591.
Chockalingam, K., Chen, Z. L., Katzenellenbogen, J. A., and Zhao, H. M. (2005) Directed evolution of specific receptor-ligand pairs for use in the creation of gene switches. Proc. Natl. Acad. Sci. USA 102, 5691–5696.
Alper, H., Fischer, C., Nevoigt, E., and Stephanopoulos, G. (2005) Tuning genetic control through promoter engineering. Proc. Natl. Acad. Sci. USA 102, 12678–12683.
Whitehead, N. A., Barnard, A. M. L., Slater, H., Simpson, N. J. L., and Salmond, G. P. C. (2001) Quorum-sensing in gram-negative bacteria. FEMS Microbiol. Rev. 25, 365–404.
Urbanowski, A. L., Lostroh, C. P., and Greenberg, E. P. (2004) Reversible acyl-homoserine lactone binding to purified Vibrio fischeri LuxR protein. J. Bacteriol. 186, 631–637.
Gilbert, W., and Muller-Hill, B. (1970) The lactose repressor. In The Lactose Operon (Beckwith, J. and Zipser, D., Eds.), Cold Spring Harbor Lab Press, Plainview, NY, pp. 93–109.
Sayut, D. J., Niu, Y., and Sun, L. (2009) Construction and enhancement of a minimal genetic AND logic gate. Appl. Environ. Microbiol. 75, 637–642.
Cadwell, R. C., and Joyce, G. F. (1994) Mutagenic PCR. PCR Methods Appl. 3, S136–S140.
Acknowledgments
This work was supported by Grant # CBET-0747728 from the National Science Foundation and Grant # IRG 93-033 from the American Cancer Society.
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Sayut, D.J., Niu, Y., Sun, L. (2011). Engineering the Logical Properties of a Genetic AND Gate. In: Wang, P. (eds) Nanoscale Biocatalysis. Methods in Molecular Biology, vol 743. Humana Press. https://doi.org/10.1007/978-1-61779-132-1_14
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DOI: https://doi.org/10.1007/978-1-61779-132-1_14
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