IGEM:Tsinghua/2007/Projects/Celcuit

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Standards in engineering cell-cell communications.
When engineering prokaryotic cell networks, one of the most difficulties is isolating signals. For one, it's necessary to get various kinds of signals to allow specific communication. In the widely-used quorum-sensing system, the quorum-sensing signal is excreted out of the cells, which in space broadcasts an identical signal to every recipient cell. In spite of the concentration gradient, that means cells with same receptor will be triggered simultaneously. And when trying to engineering a more complex system, the freedom of design is severely limited by available kinds of quorum-sensing signal producers and receptors. But if a framework allows one signal being isolated from another, it will break the those limits and allow different cells being triggered specifically. For another, 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 different combinations of signals. While the excreted broadcasting prokaryotic signals are unable to work in this way because they share the same medium or space. 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 relaxases can transport fusion proteins to recipient cells during conjugation, 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 DNA binding proteins are chosen as the transport for cell-cell signals during conjugation. Though extensively studied, the detailed mechanism of conjugation is still controversial. A popular theory postulates that a single strand DNA of host cell is carried to receptive cell by relaxase and then induce rolling replication. Some other works, however, do not support this model. Thus, we chose two kind of DNA binding proteins, including double strand DNA binding protein and single strand DNA binding protein. In both cases, several protein binding sites can be integrated in one conjugation plasmid to allow the transport of multiple copies of the signals to the recipient cells in order to amplify the effecting. 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 can be easily fused to the DNA-binding transport with pre-constructed system, allowing parallel engineering of many different pairs of signal producer and receptor. Third, the protein signal is produced in donor cells and degraded by the receptor cells. This allows reutilization of the same signals and indicates that the cellular network can be reset to the initial condition for reuse. At last, it is facile to find various kinds of protein signal producer/receptor pairs from two hybrid systems.

Prokaryotic two 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.

Flowchart
The initial part of the project pursuits the verification of relaxase and C1 transport.