Revision as of 22:47, 27 September 2016

Alverno iGEM 2016

Our Project

How can we keep genes from interfering

with each other in synthetic DNA circuits?

About This Project

Building complex biological systems with many genes requires isolating genes. Active genes can cause nearby DNA to become supercoiled, leading to unpredictable behavior of synthetic biology systems. We will test if DNA clamps (made from DNA-binding proteins) placed between genes can stop this interference. If this project succeeds, it will allow bioengineers to build more predictable genetic circuits.

What is the context of this research?

Programming cells often requires building "circuits" of several genes together on the same piece of DNA. Bioengineers have observed that when two genes are placed next to each other, they often unexpectedly interfere with each other’s expression in an unexpectedly orientation-dependent manner.

Nobody knows with certainty what causes this genetic crosstalk, but one promising theory involves DNA supercoiling. The transcription of DNA into RNA, the transcription process introduces supercoils, similar to kinks in a tightly-wound phone cord. Supercoils directly affect the expression of genes, turning them on or off depending on the direction of the supercoil.

What is the significance of this project?

If successful, a DNA-binding, gene- isolating clamp could be used in any multi-gene circuit assembly, making multi-gene assemblies more predictable and their assembly much more efficient.

This is particularly relevant when engineering metabolic pathways to produce chemicals like methanol, insulin, or antibiotics, where circuits of many genes are routinely constructed. The physical layout of these circuits can unpredictably affect production of the desired output by several orders of magnitude, so large engineered metabolic pathways must typically be hand-tuned or have many configurations screened for activity. By making gene expression more predictable, our results could greatly improve the predictability (and, therefore, designability) of large gene circuits for metabolic engineering.

Potential Applications and Implications:

If successful, a dCas9-based isolating clamp could be used in any multi-gene circuit assembly, making multi-gene assemblies more predictable and their assembly much more efficient.

This is particularly relevant in metabolic engineering, where circuits of many genes are routinely constructed. The physical layout of these circuits can unpredictably affect production of output by several orders of magnitude[4], so large engineered metabolic pathways must typically be hand-tuned or have many configurations screened for activity. By making gene expression more predictable, our results could greatly improve the predictability (and, therefore, designability) of large gene circuits for metabolic engineering.

References:

1. Yeung, E. 2016. Reverse Engineering and Quantifying Context Effects in Synthetic Gene Networks [dissertation]. [Caltech (CA)]: Caltech.

2. Tsao, Y., Wu, H., Liu, L. F. 1989. Transcription-Driven Supercoiling of DNA: Direct Biochemical Evidence from In Vitro Studies. Cell. 56:111-118.

3. Hanafi, E.D., Bossi, L. 2000. Activation and silencing of leu-500 promoter by transcription-induced DNA supercoiling in the Salmonella chromosome. Mol Microbiol. 37(3):583-94.

4. Smanski, M. J., Bhatia, S., Zhao, D., Park, Y., Woodruff, L. B. A., et al. 2014. Functional optimization of gene clusters by combinatorial design and assembly. Nature Biotechnology. 32:1241-1249.