Team:NCTU Formosa/Project/Summary





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Summary When fresh food display in a supermarket, it is necessary to detect if bacteria contaminate food or not. But it is hard to design an economical mechanism that can count the population of invading bacteria at real time and control the time of storage. To overcome this problem, our team design a genetic circuit in in E. coli that has counter and timer functions. The engineered E.coli works as a referee that can report us the situation of food using fluorescent proteins with different colors. Green fluorescent light means the referee system is turn on and environment is safe; yellow fluorescent light means the system lose freshness; and red fluorescent light indicates the storage time is up or has bacteria contamination, the environment become dangerous! We used LuxI/LuxR quorum sensing system in bacteria to design the bio-counter that can report the population of invading bacteria. Quorum sensing is a bacterial communication system that allows bacteria to sense their population density through the diffusion of a chemical signal, the concentration of acyl-homoserine lactone (AHL). As the bacteria grow, the concentration of AHL increases and diffuses within the media. When the concentration of AHL reaches a threshold level, it binds the N-terminal domain of a LuxR protein. Then the LuxR/AHL complex binds the lux box within the luxPR promoter to activate transcription of the downstream red fluorescent protein (RFP). Because the concentration of luxI protein determine how fast the AHL be produced, the counter sensitivity can be controlled by the expression level of luxI protein, that is determined by the strength of TetR repressible promoter. If external bacteria invade the system, the extra AHL produced by them will induce the RFP translation to warn us the contamination. The second function of bacterial referee is a controllable bio-timer. The concentration of lactose control how long the timer work. When added lactose is consumed by E. coli, the RFP start to translate and green fluorescent protein (GFP) is degraded. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. The red color in the final step warns us that time’s up and the product lose freshness. A key feature of the bio-timer is that make the longer timing function up to 5~8 hrs. And for the environment safety, we design a suicide system so that the E. coli would kill itself after finishing their function. <a href="http://2009.igem.org/Image:Summaryimg.png"><img src="http://2009.igem.org/wiki/images/c/cb/Summaryimg.png" ></a> Fig. 1 The bacterial referee system works as an OR gate that can integrate temporal and environmental signals. The inputs of the modular genetic OR gate are the concentration of lactose and acyl-homoserine lactone (AHL). The chemicals represent the signals from timer and counter functions. The output (RFP) of the OR gate is ON when one of the input signals is ON. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step.

We designed a modular genetic OR gate whose inputs are the lactose and acyl-homoserine lactone (AHL) to integrate the signals from timer and counter functions (Figure 1). The output (RFP) of the OR gate is ON when one of the input signals is ON. Besides, a memory system that is developed by KULeuven is applied in our referee system to memory that the inducer (lactose) has been added or not. The memory system maintains media colorless until the lactose is added.