IGEM:Tsinghua/2007/Projects/Celcuit

Celcuit

Introduction

Standards in engineering cell-cell communications.

In generally, when engineering prokaryotic cell networks, one of the most difficulties is to isolate signals. On one hand, in the widely-used quorum-sensing system, the quorum-sensing signal is excreted out of the cells, which in spite the concentration gradient in space broadcasts an identical signal to every recipient cell. Therefore, cells with same receptors will be triggered simultaneously. And when trying to engineering a more complex system, the efforts are generally limited by available kinds of signal producers and receptors. On the other hand, space isolation is equally important in engineering. Take the isolated wire cable as an example, although all the wires utilized identical electric current as the medium for signal transport, the isolation allows identical electric current in different wires to generate various combinations of signals. While the excreted broadcasting prokaryotic signals are unable to work in this way. Therefore, those requirements bring the problem posed in the field of bioengineering into a general topic in engineering cell-cell communications. It's necessary to establish a system with standards in cell-cell communication with expected isolation and variance in the kinds of signals.

Cell-cell communication platform can serve as the framework of cellular circuits.

Just take the simple case. If a system allows one cell to deliver specific signal to a specific group of cells, any complicated cellular signal systems can be built up with combinations of a proper number of such systems. It will be also feasible to construct a logical network with such framework.

Conjugation can perform as cell-cell specific signal pathway.

It is reported that during conjugation, relaxases can transport fusion proteins to recipient cells, such as Cre. Since relaxases are DNA binding proteins, we further postulated that other DNA binding proteins might also be transported into the recipient cells, which would be a potential useful phenomenon in engineering cell-cell communication. Therefore, both the relaxases and the lambda C1 proteins are chosen as the transport for cell-cell signals during conjugation. Especially, several C1 binding sites can be integrated in one conjugation plasmid allowing transport of multiple copies of the signals to amplify the signals' effects. The conjugation system has many advantages. First, the signals are transferred from one cell to another in a point-to-point manner, rather than the excreted broadcast way. Second, protein signals that can be easily fused to the transport can be easily engineered. Third, the protein signal is produced in donor cells and degraded by the receptor cells, which allow reutilization of the same signals. This means that the cellular network can be reset to the initial condition. At last, it is facile to find various kinds of protein signals.

Prokaryotic tow hybrid system is a large pool of specific signals.

During the last two decades, at least three prokaryotic two hybrid system has been established. If the two hybrid system is integrated with the cell-cell signal transport system, they can provide a large pool for specific signals in cell-cell communications.

Strains & Plasmids

Strains

Strain Source E. coli TOP10 Transgen biotech company, Beijing. E. coli S17-1 Prof. Chen's lab collection E. coli JM109 Prof. Chen's lab collection

Plasmids

Plasmid Source pEASY-Blunt Transgen biotech company, Beijing. pBBR1MCS Prof. Chen's lab collection pBluescript II SK(-) Prof. Chen's lab collection pMD18-T Takara, Japan. pLZZGPp Prof. Chen's lab collection

Flowchart

The initial part of the project pursuits the verification of relaxase and C1

transport.

Eperiment Records

Thread 1: Celcuit carrier plasmids.

Nubmer	Date	Inherit	Chart Index	Notes	Results	Plan	Expected Time	Operator
1	6-20	-	1

Thread 2: Celcuit reporter plasmids.

Nubmer	Date	Inherit	Chart Index	Notes	Results	Plan	Expected Time	Operator

*Zhenyu Shi contributed to the idea of conjugation transport signals, the experiment plan and management of this project.

*Xinyu Zhao contributed to the FLP/FRT switch idea about turning on EGFP.