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. Many influencing factors have been identified, including the recombinant plasmid copy number and the competition of translational resources between foreign genes and the bacterial genome. In order to minimize the unpredictable disturbances and stabilize 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 decide to integrate the typical control model with three nodes into our circuit. This three-module feedforward loop would act as the core foundation of our system, in which, orthogonal 16s rRNA will be a constant output through repressive and stimulative interactions. 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 is important in 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 (Known as Resource Competition). Therefore, an additional set of ribosome systems that are orthogonal to the natural ribosomes from the host 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 exciting 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

By separating the ribosome pool from host reserves, expression of proteins under correspondent orthogonal ribosome binding sites would ideally minimize the inevitable translational competition. The specific output(desired protein expression level) could further be controlled by the feedforward loop upon the binding of external AHL molecules, the only input for LuxR protein. The complex formed accordingly activates the downstream gene expression in a predictable manner, which may hopefully boost the success rate of intricate circuit designs and propel the overall development of synthetic biology.