Team:OUC-China/Description

Team OUC-China: Main

Overview

When we started

The genome contains commands which help the cell to realize some basic functions like eat, reproduce, communicate, move and excrete. Cells also can be programmed by exogenous DNA which have new commands that guide the cell to perform new artificial tasks and aims in parallel. As we all know, synthetic biology developed very fast in recent years. One of the most important things that helps us to build our fantastic dream in synthetic biology is to develop small modular genetic parts. By doing, the whole circuit will become more flexible and easy to operate. Genetic engineering is entering a new era, where microorganisms can be programmed by using synthetic constructs. More and more toolbox of modular genetic parts is designed to interface with the control of cellular processes. As we all know, the well-designed modular parts need to be assembled into biological circuits or pathways easily and different parts usually have different functions. The part created to regulate downstream gene expression is common and useful.

The existing strategies

To meet different needs, scientists have already created a lot of genetic parts based on three levels, DNA, RNA and protein. Among those three levels, post- transcriptional regulation plays a main role of gene expression. Post-translational regulation refers to the control of the levels of active protein. There are several forms. It is performed either by means of reversible events (post-translational modifications, such as phosphorylation or sequestration) or by means of irreversible events (proteolysis). To solve some current problems, more and more methods have been created based on post-translational regulation because of flexibility and practicability. The more useful toolkits we design, the more possibilities we can have.

Some methods have been created like riboswitch. A riboswitch is a regulatory segment of a messenger RNA molecule that binds a small molecule, resulting in a change in production of the proteins encoded by the mRNA. Thus, a mRNA that contains a riboswitch is directly involved in regulating its own activity, in response to the concentrations of its effector molecule. It's not only a test system, but also a way to open or close the expression of downstream genes.

Small RNA based repressor or activator is also an example. They employed a cis-repressive RNA (crRNA) element that masks the ribosome binding site (RBS) of a reporter gene through base pairing. An ectopically expressed trans-activating RNA was then designed to compete the binding with crRNA and thus to unmask RBS and restore translation.

Some problems of current methods

But some of them may have weaknesses in design. For small RNA, if the crRNA is perfectly paired with RBS, the transactivating RNA cannot compete the binding with crRNA. This may cause the low efficiency of opening this kind of switches. As for toehold switch, it is not hard to find out that each switch needs to be individually designed. At the same time, this may cause insertion of undesirable foreign sequences.

This year, our new alternative regulatory method is the ‘MiniToe Family’, which is a new toolkit based on csy4 produced by OUC-China.

Our journey

Design a new miniToe structure based on Csy4

We have test the system and it works well

The model by dry lab has showed the bigger possibility by miniToe

Design a small library based on two ways to regulate the gene expression in gradient:

1.The Csy4 mutants

2.The hairpin mutants

Test the miniToe family and it works well

Build more models for this system which help us in polycistron

Apply miniToe family to polycistrons

Use motility detection system to test and verify the function of miniToe. ( an important application for our system! )
(此处是流程图！)