Team:OUC-China/Partnership




Overview


This year, our team carried out a project inspired by biocomputer and aimed at building its basic elements-logic gates by using toehold and 3WJ. To develop the spirit of cooperation, we hope to find a relevant team for partnership. Fortunately, Worldshaper-Shanghai contacted us and told us that their project is about visual detection with toehold switch. After a deep exchange and discussion throughout the whole season, we helped them in many aspects of the project and promoted the improvement and progress of their project. Meanwhile, communication also enables us to have a more comprehensive and better understanding of Toehold.




Design


After determining the project of RNA switch and logic gate, we posted the message to find the partnership on social media. Afterward, Worldshaper-Shanghai which is consisted of senior high school students contacted us through WeChat, because both of our projects are partially related to toehold. They wanted to use toehold to test whether the samples that they collected have the specific segments of the prostate cancer gene. According to the test results, they can diagnose whether a person has cancer or not. They have a very wonderful plan, but as senior high school students, their knowledge about toehold is deficient. They did not know the details about how the toehold work and how to design a toehold to satisfy their requirements. As we all know, “There is no good building with a poor foundation”. A good knowledge base is a key to success. To enrich their knowledge about toehold and help them complete their project, we shared many useful references with them, such as Green, A. A., Silver.et al., 2014[1], Soo-Jung Kim, Matthew Leong.et al., 2019 [2] and so on. Meanwhile, we imparted our own experience to them so that they can reduce mistakes.

They had a great harvest through this communication. The experience and the literature we shared had a significant impact on them. These materials greatly enriched their knowledge of toehold, such as the specific roles of each part of toehold, the length of each part, the influence of different length on the performance of toehold, how to design their toehold switch and so on. With our help, Worldshaper-Shanghai knew toehold switch more thoroughly, and they began to design their toehold switch independently.




Model


They designed toehold following the methods in the literature we shared. They defied the switch loop size is 15nt to 18nt, the binding length is 12nt and the length of stem unwound is 14nt. Proficiency in some software can greatly simplify the works. The NUPACK is a convenient tool to design and analyze RNA structure. In order to simplify their work, they wanted to use NUPACK to generate the required Toehold sequence. However, they didn’t know how to code in NUPACK.

NUPACK has also been used many times in our project to design different logic gate sequences. So we shared the code that we wrote for them and told them how to write the code in NUPACK. Using the code, they can get the whole sequence of toehold switch when they input the sequence which is complementary to trigger. The application of NUPACK greatly reduced the workload.




HP


With the experience of conceiving the project and designing toehold, Worldshaper-Shanghai had a better understanding of toehold and they planned to write a design guide of toehold for novices. This design guide will help the novices master the design method faster. At the same time, it will also in favor of the widespread use of toehold. In order to ensure that this guide is scientific and accurate, they invited us to modify their design guide. We put forward some suggestions on the modification of the vague definition and perfected the details.


Figure 1. Design guide of toehold switch





Wet Lab


Seriously affected by the epidemic, Worldshaper-Shanghai can’t begin their laboratory work at first. But the terrible situation does not restrain their enthusiasm for experimentation. So, they have made some experimental plans and searched for some protocols.

As a project of diagnosing prostate cancer, convenience and fast are first cause should be considered. According to their initial design, they planned to amplify the target gene by PCR. However, PCR has a complicated process, which means it’s not convenient to use and time-consuming. For the kit they designed can be applied to a more general environment, we introduced Recombinase Polymerase Amplification (RPA) to them, which can work efficiently at the same temperature. In order to help them understand the RPA, we shared the relevant reference with them. This useful idea was added to their future work, which made their easy and quick diagnosis go further.




Education


In order to disseminate synthetic biology knowledge, we have published a total of ten editions of comics on OUCiGEM WeChat platform, which were read by thousands of people. Worldshaper-Shanghai also saw the comics that we have published. They thought that our science cartoons are very easy to understand and fascinating. Inspired by the success of our self-media platform, they also wanted to tell their own stories in the form of cartoons, to popularize the knowledge of synthetic biology to the general public. They cooperated with KEYSTONE 2020 iGEM team to design a game application that can introduce basic biology ideas through scene play.





References

[1] Green, A. A., Silver, P. A., Collins, J. J., and Yin, P. (2014) toehold switches: de-novo-designed regulators of gene expression. Cell 159, 925– 939, DOI: 10.1016/j.cell.2014.10.002

[2] Soo-Jung Kim, Matthew Leong, Matthew B. Amrofell, Young Je Lee, and Tae Seok Moon ACS Synthetic Biology 2019 8 (3), 601-605.DOI: 10.1021/acssynbio.8b00488