Team:ZJU-China/Project

Project Description

The assembly of genetic circuits is a huge obstacle between designs and achievements in synthetic biology. Many well-known methods, from traditional restriction digestion & ligation, 3A assembly to Gibson assembly, aim to overcome the difficulties but unfortunately get respective defects. This year, ZJU-CHINA seeks to build a gene-insertion system in bacterial chromosome, "GeneSocket". Clearly different from the in vitro constructing methods mentioned before, GeneSocket, which can be easily combined with existing in vitro methods, makes gene expression more accurate, stable and controllable by assembling genetic elements in chromosome directly.

Two core methods, lambda red recombination and recombinase-based bistable switch are applied to build our GeneSocket. Lambda red recombination is a widely used, efficient recombination system in prokaryotes. Recombinase-based bistable switch is relatively more stable and easier than transcription factor regulated bistable switch modules. Both the two are the best choices for achieving the characteristics of GeneSocket.

We hope that by using GeneSocket, synthetic biologists can turn their theoretical design into reality faster and better. We want to lead to the revolution in techniques of synthetic biology!

• A Design
• A Method
• A Product

Why circuit construction?

Genetic regulatory circuits are artificially-designed gene clusters constructed by disparate genetic elements, which can produce novel genetic function according to people’s desire. Able to be modeled and simulated in silico, genetic regulatory circuits can be either qualitatively or quantitatively examined, with its function even able to be predicted. Sound construction of circuits has been one of the crucial parts of synthetic biology, which has been taking people great efforts to perfect.

Why on chromosome?

Nowadays, modern techniques have enabled the construction of more complicated and large-capacity genetic systems. However, most of the genetic circuits are constructed on plasmids, which, with the increasing of complexity, has brought about incremental uncertainty and unpredictability. This indeterminacy is mainly generated by plasmid loss, allele inactivation, copy number variability or plasmid-associated metabolic burden[1]-[4]. To obtain optimal performance in certain microbial host, rounds of examination and troubleshooting may be needed. Nevertheless, with genetic regulatory circuits constructed on microbial chromosome or on bacterial artificial chromosome (BAC), more robust systems can be obtained.

Why GeneSocket?

With more and more complicated genetic circuits designed, more efficient, time-saving and inexpensive methods are needed to bring the design into reality. Many well-known methods like traditional restriction digestion and ligation, 3A assembly and Gibson assembly all aim to overcome the difficulties of gene assembly while problems like cumbersome steps, low cost performance, lots of time consuming do not receive quite effective solutions. The new gene-insertion method we build, which we call GeneSocket, is able to assembly genetic elements in vivo efficiently, with reporters easy to be identified, and simple isolation methods. We hope that it can become another choice for researchers in future.

References

[1]Santos, C. N. S. & Yoshikuni, Y. Engineering complex biological systems in bacteria through recombinase-assisted genome engineering. Nature Protocols 9, 1320-1336, doi:10.1038/nprot.2014.084 (2014).

[2]Tyo, K.E., Ajikumar, P.K. & Stephanopoulos, G. Stabilized gene duplication enables long-term selection-free heterologous pathway expression. Nat. Biotechnol. 27, 760–765 (2009).

[3]Bentley, W.E. & Quiroga, O.E. Investigation of subpopulation heterogeneity and plasmid stability in recombinant Escherichia colivia a simple segregated model. Biotechnol. Bioeng. 42, 222–234 (1993).

[4]Paulsson, J. & Ehrenberg, M. Noise in a minimal regulatory network: plasmid copy number control. Q. Rev. Biophys. 34, 1–59 (2001).

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