Refinement
Add New Documentation to An Existing Part
We provided additional experimental data on the sensitivity of the tryptophan-sensitive promoter pTrpR (Part: BBa_K191007) to different concentrations of tryptophan.
From literature research, we know that Escherichia coli has its own TrpR-pTrp system to sense tryptophan concentrations, which regulates the tryptophan biosynthesis pathway. Therefore, we cloned TrpR into the A module and TrpO into the R module, followed by the sfGFP reporter gene. In the presence of tryptophan, TrpR binds to TrpO, preventing transcription and translation of the fluorescent protein downstream of TrpO. In the absence of tryptophan, this pathway is activated, allowing downstream gene expression and the production of the fluorescent protein.
- A Module: J23119 - TrpR - B1006 - pUC57 (Kana)
- R (Reporter assay) Module: pTrpRO - sfGFP - B1005 - p15A (Chl)
We synthesized gene fragments with standardized interfaces and assembled them into the designed standard vectors using Golden Gate assembly. The plasmids were transformed into DH5α, and three single colonies were selected and sent to Azenta for sequencing, while the remaining cultures were preserved with 20% glycerol.
From the preserved strains, we streaked them on LB plates with the corresponding antibiotic to isolate single colonies. Three single colonies were selected and inoculated into LB medium with the appropriate antibiotic for overnight culture. The next day, the culture was diluted to the logarithmic growth phase, and different concentrations of tryptophan were added. After 16 hours of incubation, 150 μL of the culture was transferred to a TECAN plate reader to measure the sfGFP fluorescence signal and OD values.
Table 1. Response of the TrpR-pTrp system to different concentrations of tryptophan solution.
In this experiment, we used the inhibitory Hill equation model to fit the relationship between tryptophan concentration and the biosensor response. The results showed that our system exhibited the highest sensitivity to tryptophan at a concentration of 0.03 mol·L⁻¹, with a wide detection range that could detect tryptophan from very low to high concentrations. Additionally, we found that the tryptophan binding process was nearly non-cooperative. This means that the binding of tryptophan molecules does not significantly affect the binding of subsequent molecules, resulting in a relatively flat response curve.
Figure 1. Hill equation fitting curve of the TrpR-pTrp system response to different concentrations of tryptophan solution.
You can find more information at the following links: parts, BBa_K191007.
Innovation
Open-Source POCT Hardware with Biosensor Fluorescence: A Practical and Collaborative Innovation
We have created an open-source, modular Point-of-Care Testing (POCT) hardware system that uses biosensor-based fluorescence for biomarker detection. Our system is designed to be adaptable, allowing for easy customization to detect a variety of biomarkers, making it a valuable tool for both practical use and as a contribution to the iGEM community. By incorporating multiple interchangeable modules, such as optical, fluidic, and temperature control, the design ensures broad compatibility for future applications.
New Composite Part
To detect a wider range of biomarkers, we optimized and improved the plasmid system by introducing a GoldenGate interface at key sites in both the A module and R module. This enhancement will significantly increase the system's flexibility, making it easier to integrate additional biological components in the future, thereby expanding the system's capability to detect multiple biomarkers.
You can find more information at the following links: Parts, BBa_K5460000.
Empowerment
Future Adaptability with Standardized Biosensors
In the future, this POCT hardware system will allow for easy biomarker detection by simply replacing the microfluidic chip with one containing the appropriate biosensor for the target biomarkers. The system’s modular and adaptable design ensures that the hardware remains consistent while only the biosensor needs to be modified for different applications. Moreover, the standardized design of these biosensors can be referenced from the open-source documentation provided by our bioengineering team. This ensures that future iGEM teams can easily adapt and develop new biomarkers within the existing hardware framework, promoting further innovation and collaboration in the community.
Future Adaptability with Hardware
This open-source POCT system lowers the barrier for iGEM teams to develop their own diagnostic tools using biosensors, encouraging innovation and facilitating collaboration within the community. With comprehensive documentation provided, we aim to make this system easily replicable and modifiable for future teams. Our goal is to contribute both practically, by offering a flexible and efficient testing tool, and collaboratively, by empowering the iGEM community with a robust, open-source foundation.
We invite everyone to explore this system at the iGEM Jamboree in Paris, where we will showcase its structure, functionality, and potential for future use.
