Part:BBa_K5080001
AHL molecule-activated promoter for testing with real milk samples.
This is a reporter assay activated by AHL molecules. We have integrated the pLux promoter with mVenusNB, a fluorescent protein, to engineer this construct.. It must be used in conjunction with BBa_K5080000, which expresses the LuxR protein responsible for binding the small molecule AHL in the environment. Once LuxR complexes with different small molecules, it can activate pLux, thereby triggering the expression of downstream fluorescent proteins. The activation strength of pLux by AHL-LuxR complexes of varying concentrations is different, so we can determine the molecular concentration of AHL in the system based on the intensity of the fluorescence signal.
A significant amount of work 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 and the pLux promoter, and placed them on two distinct plasmids, creating a dual-plasmid system that responds exclusively to AHL molecules, and tested this system's response to real milk samples. By adjusting the promoter of LuxR, we have performed parameter optimization for the entire system.
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.
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.
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.
Reference:
[1] LuxR- and LuxI-Type Quorum-Sensing Circuits Are Prevalent in Members of the Populus deltoides Microbiome. mBio, Available at: https://doi.org/10.1128/mBio.01722-15.
[2] Odeyemi, O.A., Alegbeleye, O.O., Strateva, M. and Stratev, D. (2020) Understanding spoilage microbial community and spoilage mechanisms in foods of animal origin. Comprehensive Reviews in Food Science and Food Safety, 19, pp. 311-331. doi: 10.1111/1541-4337.12526.
[3] IIT Kharagpur iGEM Team (2015) Project Overview. Available at: https://2015.igem.org/Team:IIT_Kharagpur/Project.
[4] Tokyo Tech iGEM Team (2016) AHL Assay: AHL Reporter Assay. Available at: https://2016.igem.org/Team:Tokyo_Tech/AHL_Assay/AHL_Reporter_Assay.
[5] Michigan iGEM Team (2019) Project Overview. Available at: https://2019.igem.org/Team:Michigan/Project.
[6] GCGS China iGEM Team (2021) Project Description. Available at: https://2021.igem.org/Team:GCGS_China/Description.
[7] Kim, J., Twede, D. and Lichty, J. (1997) 'Consumer attitudes about open dating techniques for packaged foods and over-the-counter drugs', Journal of Food Products Marketing, 4(1), pp. 17-30. doi: 10.1300/J038v04n01_03.
[8] United States Food and Drug Administration (2008) Foodborne Illness-Causing Organisms in the U.S.―What You Need to Know. Available at: https://www.fda.gov/food/foodborne-pathogens/foodborne-illness-causing-organisms-us-what-you-need-know.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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