Team:NCTU Formosa/WetLab/Counter





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Result - Counter

Outline We designed an economical mechanism that can count the population of invading bacteria at real time with LuxI/LuxR device. The counter function in engineered E.coli can remind us the situation of environment by different color fluorescent proteins. Green fluorescent light means the counter system is turn on and environment is safe; yellow fluorescent light means the system lose freshness; and red fluorescent light indicates system has bacteria contamination and the environment become dangerous! <a href="http://2009.igem.org/Image:Counter01.png" ><img src="http://2009.igem.org/wiki/images/4/47/Counter01.png" border="0"></a> Component Descriptions

<ol> <li>LuxI protein (BBa_C0061) is the synthase that convert S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). 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 Plux/cIIp22 (BBa_K145150) promoter to activate transcription of the downstream red fluorescent protein (RFP).</li> <li>LuxR (BBa_C0062) is transcript continuously by the constitutive promoter (BBa_J23106), and waits for working with AHL. This design can control the concentration of LuxR protein in a steady phase, and the concentration of AHL/LuxR complex is only affected by the concentration of LuxI protein (BBa_C0061).</li> <li>Plux/cIIp22 (BBa_K145150) promoter is actived by AHL/LuxR complex and repressed by cIIP22 (BBa_C0053) protein. 4.	Since the expression level of LuxI protein is determined by the strength of the PtetR promoter (BBa_C0040), the counter sensitivity is determined by the strength of the PtetR promoter (BBa_C0040).</li> <li>Since the expression level of LuxI protein is determined by the strength of the PtetR promoter (BBa_C0040), the counter sensitivity is determined by the strength of the PtetR promoter (BBa_C0040).</li> <li>Strand C is working in lactose-consumption phase only.</li> </ol>

<Strong>Principle and Mechanism</Strong> The memory system in our bacterial referee has three stages: <ol> <li>Standby phase: bacteria grow in lactose-free medium.</li> <li> Lactose-accession phase: lactose is added in medium. </li> <li> Lactose-consumption phase: bacteria eat all lactose, and the medium does not contain lactose. </li> </ol> <ol> <li>Standby stage: bacteria grow in lactose-free medium, and the medium is colorless <a href="http://2009.igem.org/Image:Counter02.png" ><img src="http://2009.igem.org/wiki/images/5/5b/Counter02.png" border="0"></a> When bacteria grow in lactose-free medium, the LuxR and lacI protein is transcript continuatively. The LacI repressor (BBa_K091121) inactive the promoter Plac (BBa_K091110) on strand B and GFP dose not transcript,. The strand E produce cIIP22 repressor witch inhibit the promoter Plux/cIIp22 (BBa_K145150) on strand F. Therefore, mRFP ( BBa_E1010) dose not transcript, and counter system is colorless in standby phase. </li> <li>2. Lactose-accession phase: lactose is added in medium. <a href="http://2009.igem.org/Image:Counter.png" ><img src="http://2009.igem.org/wiki/images/b/be/Counter03.png" border="0"></a> When lactose is added in medium, lactose binds with lacI (BBa_K091121) repressor and releases the inhibition of B strand, so the PlacI (K091110) promoter starts transcripting GFP-LVA(BBa_K145015). Then, the medium are green in this stage. </li> <Br> <li>Sensing phase: external bacteria invade the system, and medium becomes red. <a href="http://2009.igem.org/Image:Counter04.png" ><img src="http://2009.igem.org/wiki/images/9/99/Counter04.png" border="0"></a> 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 diffusion of a chemical signal- the concentration of acyl-homoserine lactone (AHL). In this bio-counter, LuxI protein (BBa_C0061) is the synthase that convert S-adenosylmethionine (SAM) into acyl-homoserine lactone (AHL). When external bacteria invade the system, the concentration of AHL will increase and diffuse into the media. When the concentration of AHL reaches a threshold level, then AHL can bind the LuxR (BBa_C0062) protein to from the LuxR/AHL complex. This complex activate target promoter Plux/cIIp22 (BBa_K145150) to transcript downstream Red Fluorescent Protein (RFP) ( BBa_E1010) and the medium is red in this stage. </li> </ol>

Important concept 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 the PtetR promoter (BBa_C0040). Our team designs a simple and rapid protocol to generate a promoter library. This promoter library with different transcriptional strength can be built to tune the specific promoter activity for our design. The multi-faced characterization of promoter strength enabled identification of optimal response for the bio-conter. Submitted parts in this counter

<a href="http://2009.igem.org/Image:Counter05.png" ><img src="http://2009.igem.org/wiki/images/d/d3/Counter05.png" border="0"></a>