Description

ShanghaiTech 2018

Project introduction

The construction of increasingly complex genetic networks in engineered bacteria has been particularly susceptible to circuit failure, due to undesirable expressions of proteins involved. Varied factors have been identified as influencing, including the recombinant plasmid copy number and the competition of translational resources between foreign genes and the bacterial genome. In hopes of minimizing the unpredictable disturbances and stabilizing the expression of genetic circuits, a system employing feedforward control and orthogonal ribosomes is devised.

The feedforward control circuit

Inspired by the control theory that developed into the system biology, we choose to apply the typical control model of three nodes in our circuit. A three-module feedforward loop would act as the base of the system, which, through repressive and stimulative interactions, keeps a constant output of orthogonal 16s rRNA. The first module is the LuxR, which is a commonly-utilized part in synthetic biology from the quorum sensing in origin. It stimulates the second module, the STAR RNA and meanwhile the output, orthogonal ribosome. The STAR RNA would continue to trigger the third module, the negative RNA transcriptional regulator pT181, which then suppresses the LuxR expression and complete the negative feedback loop.

The three modules are orthogonal to each other and the host itself, which matters a lot in the artificial design of gene circuits.

The orthogonal ribosome

When expressed in genetically engineered bacteria, foreign proteins compete with the native proteins of the host for ribosomes, affecting the stability of the expression system, which is known as the Resource Competition. Therefore, an additional set of ribosome systems that are orthogonal to the host's natural ribosomes is particularly critical.

The basic principles of the artificial ribosome design are mutations in the SD region of the 16s rRNA so that it can only read and translate the specific mRNA. Several great outcomes have been achieved by the scientists working on this theme. We have tried to apply two editions of the orthogonal ribosome design so far. One design originates from Jason Chin's lab (https://www2.mrc-lmb.cam.ac.uk/ccsb/), the other one comes from the iGEM team of Tianjin University in 2012(https://2012.igem.org/Team:Tianjin).

Project Summary

Expression of proteins under correspondent orthogonal ribosome binding sites would then have translational competition minimized thanks to the separated ribosome pool from host reserves. The specific output level could further be controlled through the amount of external AHL molecules, thus ensuring a desirable expression yield. The predictability of circuits enabled is in the expectation of boosting the success rate of intricate circuit designs and propelling the overall development of synthetic biology.