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Artificial Cooperation System

Requirement

In order to make our system, we need introduce cell-to-cell communication mechanism. Many cell-to-cell communication mechanisms in bacteria are known, though the mechanisms of many systems are not well-understood yet. Thus we used “quorum sensing” whose mechanism is well studied. And quorum sensing is popular in synthetic biology and iGEM.

quorum sensing

Quorum Sensing (QS) refers to cell-to-cell communication systems that are used by many microorganisms to sense their local cell densities. This sensing mechanism is based on the production, secretion and detection of small signaling molecules, whose concentration correlates to the abundance of secreting microorganisms in the vicinity. When the signal concentration reaches a threshold, they know the ‘quorum’ is formed, and the communicating microorganisms undergo a coordinated change in their gene-expression profiles. As a result, they initiate complicated activities which would not have been occurred at smaller cell numbers. Secretion of virulence factors, initiation of biofilm formation, sporulation, competence, mating, root nodulation, bioluminescence, and production of secondary metabolites are the examples for the activities mentioned above.

There are two regulatory genes involved in QS, I and R genes. The I gene produces I protein and directs the synthesis of an N-acyl homoserine lactone (AHL). AHL is bacteria-specific and gene-specific signal molecule. On the other hand, R protein, which is expressed by R gene, binds to the synthesized AHL signal molecule. The AHL and R protein complex acts as a transcription factor.

In synthetic biology field or iGEM, lux, las, and rhl have been used frequently. ‘‘luxI’’, ‘‘lasI’’ and ‘‘‘‘rhlI’’’’ gene synthesize N-(3-oxohexanoyl) homoserine lactone(3OC6HSL), 3-oxododecanoyl- homoserine lactone (3OC12HSL) and butanoyl- homoserine lactone (C4HSL) ,respectively. LuxR, LasR and RhlR receptor have been synthesized and can bind to 3OC6HSL, 3OC12HSL and C4HSL, respectively.

In QS, not only activation promoters but also repression promoters have been used. The fact that LuxR and TraR promoter can function as both activation and repression has already been published in a paper*. We have characterized luxR promoter in order to confirm this feature of LuxR protein.

• Conversion of the Vibrio fischeri Transcriptional Activator, LuxR, to a Repressor