Our project was designed to be compatible with the conventional procedure and replace the traditional machinery as a next-generation approach. Therefore we took the entire procedure into consideration for the project to act as a whole in real circumstances during implementation.
The main end users of our project are scientific researchers and third-party environmental governance enterprises, similar to the users of conventional tools. We envision them using our project by ordering biosensor strains just like how they normally order needed equipment. However, the specific range of users for our biosensor will be larger because the biosensor is more affordable. This is proven by our human practices since we found that middle- or small-sized enterprises do not possess the economic capacity to use more accurate technologies such as ICP-MS. This means that smaller enterprises will benefit a lot from our biosensor since their costs will be reduced. For environmental enterprises and researchers, our biosensor is projected to possess the benefits of being not only cost-effective but also environment-friendly, portable, and efficient, while having satisfying accuracy, therefore offering a next-generation approach to pollution detection.
The usual full working procedure for pollution detection contains three major steps: sampling, experimentation, and data analysis. Our major focus, of course, is on experimentation. But our project encompasses all three of the steps. The following is how our work is related to the steps.
For sampling, in traditional methodologies, the machines are usually large and impossible to bring to the wild for testing. Although our strains at the current stage also cannot be brought out of the laboratory for safety considerations, after our product is fully developed it might be possible to conduct wild sampling and simply experiment immediately after sampling. This will be done using our specially designed hardware prototype. It is a biosafety box that could be used in sampling under natural conditions. The box has two parts: one which could be used to place sampling tubes, and another which contains wells for strain cultivation and experimentation. It also has a screw-compatible lid to prevent strain leakage. The users can cultivate strains in this box and directly take the box with them during wild experimentation. Then they can simply take the samples in tubes, put them in the box, carry out the pipetting, wait for color or luminescence to show, and compare the results with a fixed set of heavy metal concentrations-reporter concentrations correspondences. We hope that it could be used in the future when the biosafety of our biosensor has been confirmed.
The box prototype was 3D-modeled and printed with heat-insulated and corrosion-resistant materials. Its body is composed of two parts: 16 large wells for holding experimental tubes, and 49 small ones for strain cultivation. The lid also has two parts. The one fitting with the part of smaller wells directly presses against the plane on which the wells are based to prevent leakage, while the one over the larger wells left enough space for the experimental tubes.
The experimentation process will be carried out simply by combining the strain solution and the samples. In order for the samples to be used, we first have to gain official approval. In China, relevant laws and regulations require that new methodologies must undergo characterization independently by several laboratories to confirm their stability and accuracy (roughly similar to iGEM’s Interlab project) to confirm the sensor’s accuracy and stability, which has not been achieved so far. In this respect, the results of our experimentation have convinced us that the methodology of high-throughput experimentation, which we have adopted, is effective. We will continue improving our biosensors in order to meet the regulations. Currently, we have confirmed three families of parts that can be based on for future continuations of our project. More information relating to this can be found on the Model page, and more information on our current experimental progress can be found on the Results page and the Part pages.
Our current project still requires further improvement since the detection limits of most of our strains remain around the mM level, indicating poor sensitivity. Their stability is also worrying. However, we have already demonstrated the feasibility of our approach, as the original PIII-csoR system wasn’t copper sensitive at all, while now we have found functioning variants. This brings us confidence that, given further time and resources, we can successfully improve the current variants to make them work as expected.
We will continue this project in the next months. We have also successfully received project approval for the 2022 National Innovation and Entrepreneurship Plan for College Students, mentored by experts from CRAES.
As for data analysis, we have also constructed a model for predicting the performances of our parts based on related parameters. This could guide the construction of data analysis standards for more individual types of heavy metals, and could be used in the future construction of a database for the characterization of different heavy metals. Due to the lack of time for detailed characterizations, we did not carry out this work, but in the following project execution, we will do this based on our improved biosensors.
We have also considered the risk of our GMOs carefully. Although through literature review and expert interviews we decided that a biosafety circuit is unnecessary, more experimental evidence is needed to confirm that leakage will not cause environmental threats. In addition, we might want the full development of our project to encompass natural sampling, therefore requiring not only biosafety but also stability of the bacteria under pressure. Previously we have even considered using suicidal modules to prevent leakage, which led to our investigation and work on the Doc toxin (now we registered it as BBa_K4494197). However, we eventually decided that it is unnecessary since our parts do not offer the GMOs a greater opportunity to survive, and therefore leave them easily outcompeted in natural selection. We have also considered if our end users are capable of preserving our strains in conditions with enough biosecurity. We received positive results regarding this during our interviews since we found that even medium- or small-sized enterprises have bio-laboratories that are capable of holding bacteria safely.
After our approach is officially accepted, we need to commercialize it. As for this, our stakeholder interviews confirm that related enterprises and researchers are eager to see a new generation of methodologies develop. This could also be further evidenced by the current rapid surge of synthetic biology on the market, showing a general welcome for the commercialization of new technologies.