This year, our team submitted some documents from experiments to several different parts. In addition, we have constructed a collection of composite parts of nitrate sensor system, which made preliminary explorations in expanding this system to more application areas.
More importantly, relying on the resources of iGEM, we cooperated with six teams (BNUZ-China, BUCT, Nanjing-China, NJMU-China, SUSTech_Shenzhen, SZU-China) to accomplish the Intestinal Program Reference Manual. We hope that the manual can offer guidance for future teams, providing them different perspectives to think about problems, and reminding them of some easily overlooked issues.
Characterize Existing Parts
We completed the experimental characterization of the parts (BBa_K3247005 and BBa_K1162006) and added some information of them to the corresponding parts.
The RNA thermometer has a high fold change between 25°C and 37°C. To characterize the regulatory function of the RNA thermometer, we measured the relative fluorescence of the engineering bacteria with thermometer and GFP gene at different temperature.
Figure 1. The characterization result of RNA thermometer.
For more information you can see here: BBa_K3247005
LL-37, one of the antimicrobial peptides (AMPs), could resist the spread of bacteria by neutralizing LPS. The LL-37 has a broad-spectrum antibacterial property, which means it can inhibit the growth of diverse strains. Here, we took gram-negative bacteria Salmonella Typhimurium SL1344 which can exist in the animal’s intestine as an example to verify the function of LL-37.
Figure 2. The characterization result of LL-37.
For more information you can see here: BBa_K1162006
Designing a gene circuit is relatively simple, but meeting the design requirements is extremely difficult. The main reason is that the different parts will be interfered by each other when they are assembled into a device, which will affect the function of both the device and parts. In our project, the “Detection Module” can fulfill the function we expected. However, there is still a long way to apply our design to the daily situation. On the basis of satisfying our demands, we further explored the performance of the NarXL two-component system in different circuits to expand the application area.
We constructed a collection of nitrate sensor circuits, including the combinations of different promoters and , ribosome binding sites (RBS) and the presence or absence of RiboJ. Summarizing the results, the nitrate sensor system has different performances under different conditions. But in most cases, the nitrate sensor system can maintain the function.For more information you can see here: Measurement
Intestinal Program Reference Manual
During the communication with SZU-China, we found many common problems. Later, we discovered that these problems are common in other projects related to the intestine. After communicating with other teams whose project also focused on bowel, SZU- China put forward the idea of Intestinal Program Reference Manual. Our team stands that it’s our duty and obligation to participate in it and spare no effort to make our contribution as the pioneers of this field. Finally, there are 7 teams in total to compose the manual, on the same purpose to inspire future workers in intestine-related field and summarize some easily neglected problems or issues.
After several discussions, the Intestinal Program Reference Manual was ultimately divided into three parts, including dry lab, wet lab and ethics. We undertook the responsibility to dig deep into biosafety and the approaches to prevent horizontal gene transfer which was comprised in the wet lab. In the process of finishing our part of work, we filed and arranged all the Toxin-Antitoxin systems (TA) once used in previous competition. The corresponding results are attached below. Besides, we also concluded the overall design of the two best safety projects, which had inspired us a lot. After the first version was completed, we turned to Nan Zhang-CCiC for advice and further modification, hoping to illustrate a complete template for further researchers and pass our manual down as a beacon.
We sincerely hope that the Intestinal Program Reference Manual can provide a standardized method, thus giving guidelines for a wider range of people. Just like the concept “BioBrick” in synthetic biology, different parts in the manual will function as different units in the project, having the capability of integrating into more projects. Therefore, the manual will undoubtedly have a broader application scenario.For more information you can see here:Intestinal Program Reference Manual