Difference between revisions of "Part:BBa K5080000"

 
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        <p><strong>The LuxR expression system, for use in testing real milk samples.</strong><br>This part is utilized to express the LuxR protein, serving as the regulatory protein in our milk detection system. It needs to be used in conjunction with BBa_K5080000; BBa_K5080000 expresses the LuxR protein, which is responsible for binding AHLs in the environment. After LuxR complexes with AHLs at different concentrations, it can activate pLux, thereby initiating the expression of downstream fluorescent proteins. The activation strength of pLux by AHL-LuxR complexes of varying concentrations is different, allowing us to determine the molecular concentration of AHL in the system based on the intensity of the fluorescence signal.<br>Significant research has been conducted on the Lux quorum-sensing system from Vibrio fischerii. Traditionally, the entire system was constructed on a single plasmid. This year, we have separated the two most critical components of this system: <strong>LuxR</strong> and<strong>pLux</strong>, and placed them on two distinct plasmids (corresponding to BBa_K5080001 and BBa_K5080000), creating a dual-plasmid system that responds specifically to AHL molecules, and tested this system's response to real milk samples.<br> <strong>Additionally, we tested the system's response to AHL small molecules under the control of the LuxR protein driven by J23119 and 16 other promoters listed in the iGEM part registry.</strong></p>
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LuxR and pLux form a well-established transcriptional regulatory protein and its corresponding promoter. In bacteria, once LuxR binds to AHL, the resulting complex binds to the pLux promoter region, initiating the expression of downstream genes. The concentration of AHL is related to bacterial population density, and when the concentration exceeds a certain threshold, the LuxR-AHL complex activates the expression of downstream genes. This plasmid uses the strong promoter J23119 to drive high levels of LuxR protein expression in Escherichia coli, allowing it to work with BBa_K5080001 to detect the concentration of AHL molecules in the environment.
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            <img src="https://static.igem.wiki/teams/5080/part-registry/trancription-unit-1.jpg"alt="图一" style="width: 300px; margin-right: 10px;">
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            <img src="https://static.igem.wiki/teams/5080/part-registry/trancription-unit-2.jpg"alt="图二" style="width: 300px;">
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===Usage and Biology===
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        <p><strong>Part I: Standard Sample Testing</strong><br>First, we tested the influence of different standards on this dual-plasmid system and obtained the AU-time curve. We then plotted a standard curve correlating the stabilized fluorescence values with the corresponding AHL small molecule concentrations.</p>
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<span class='h3bb'>Sequence and Features</span>
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            <img src="https://static.igem.wiki/teams/5080/part-registry/au-time.jpg" alt="图三" style="width: 400px; margin-right: 10px;">
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            <img src="https://static.igem.wiki/teams/5080/part-registry/rfu-ahl.jpg" alt="图四" style="width: 400px;">
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      <p><strong>PartII :Milk Sample Testing</strong><br>Subsequently, we tested the system's response to AHL-type small molecules produced by microbial growth in actual spoiled milk samples and obtained positive results. This verified the concept of using our system for milk testing.</p>
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===Functional Parameters===
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            <img src="https://static.igem.wiki/teams/5080/part-registry/au-time-milk.jpg" alt="图三" style="width: 400px; margin-right: 10px;">
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        <img src="https://static.igem.wiki/teams/5080/part-registry/milkzhuzhuangtu.jpg" alt="图三" style="width: 400px; margin-right: 10px;">
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        <p><strong>PartIII :High-Throughput Screening (Model Refinement)</strong><br>Ultimately, we refined the entire system by replacing the promoter sequence upstream of LuxR, which yielded an extensive dataset detailing the influence of various promoter-driven LuxR expressions on the system's response intensity. Utilizing this empirical data, we developed a predictive model to elucidate the variations observed.For detailed information regarding this model, please refer to the model page on our wiki.</p>
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          <img src="https://static.igem.wiki/teams/5080/part-registry/bar-chart-of-16-promoters.jpg" alt="图三" style="width: 400px; margin-right: 10px;">
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Revision as of 07:57, 28 September 2024

The LuxR expression system, for use in testing real milk samples.
This part is utilized to express the LuxR protein, serving as the regulatory protein in our milk detection system. It needs to be used in conjunction with BBa_K5080000; BBa_K5080000 expresses the LuxR protein, which is responsible for binding AHLs in the environment. After LuxR complexes with AHLs at different concentrations, it can activate pLux, thereby initiating the expression of downstream fluorescent proteins. The activation strength of pLux by AHL-LuxR complexes of varying concentrations is different, allowing us to determine the molecular concentration of AHL in the system based on the intensity of the fluorescence signal.
Significant research has been conducted on the Lux quorum-sensing system from Vibrio fischerii. Traditionally, the entire system was constructed on a single plasmid. This year, we have separated the two most critical components of this system: LuxR andpLux, and placed them on two distinct plasmids (corresponding to BBa_K5080001 and BBa_K5080000), creating a dual-plasmid system that responds specifically to AHL molecules, and tested this system's response to real milk samples.
Additionally, we tested the system's response to AHL small molecules under the control of the LuxR protein driven by J23119 and 16 other promoters listed in the iGEM part registry.

图一 图二

Part I: Standard Sample Testing
First, we tested the influence of different standards on this dual-plasmid system and obtained the AU-time curve. We then plotted a standard curve correlating the stabilized fluorescence values with the corresponding AHL small molecule concentrations.

图三 图四

PartII :Milk Sample Testing
Subsequently, we tested the system's response to AHL-type small molecules produced by microbial growth in actual spoiled milk samples and obtained positive results. This verified the concept of using our system for milk testing.

图三 图三

PartIII :High-Throughput Screening (Model Refinement)
Ultimately, we refined the entire system by replacing the promoter sequence upstream of LuxR, which yielded an extensive dataset detailing the influence of various promoter-driven LuxR expressions on the system's response intensity. Utilizing this empirical data, we developed a predictive model to elucidate the variations observed.For detailed information regarding this model, please refer to the model page on our wiki.

图三 图三