In order to help the future IGEM team better brainstorm and complete the competition, the team believes that it is responsible to provide constructive help and suggestions to the future team in terms of wet lab, human practice and others.

Safety Support

  We have good innovation and investigation in terms of safety. Since most of the future IGEM teams will be involved in transgenic technology, especially the team that genetically modified E. coli. Once GMOs cause GMO pollution, it has a certain impact on our living environment and other organisms. So we designed a suicide system. Our plan is to insert an antitoxin gene behind a gene that we have engineered, and then express a toxin gene in the back. Arabnose is present in the small intestine, and arabinose is not broken down by the small intestine. Therefore, when E. coli is in the small intestine, it can receive arabinose in the small intestine, express the antitoxin gene in the back, and neutralize the toxin gene expressed later, and the bacteria can survive normally. When E. coli leave the small intestine, arabinose operons that have lost arabinose regulation do not express the subsequent antitoxin gene, only the toxin gene is expressed. The bacteria complete the suicide. Toxin genes and anti-toxic genes are difficult to find, and a lot of literature needs to be investigated, but fortunately, the team solved the difficulties and found a suitable pair of genes.

  The toxin gene kid and antitoxin gene kis contained in the toxin-antitoxin system parD of plasmid R1 were selected by us as the main components of the suicide system, as shown in the following figure, we inserted a promoter specifically expressing the kis antivirus gene on the basis of the original arabinose manipulation, in the D region of the last expression of the poor isomerase, and then inserted the kis gene, expressing the P10 antitoxin protein kis ( In the presence of arabinose) insert the promoter of the kid and kis genes after the kid gene and the kid gene of the toxin protein.

  In the future, if the team doing genetic modification of E. coli can learn from the above ideas and specific genes, you can refer to some literature such as Ruiz-Echevarría, M J et al. "The kis and kid genes of the parD maintenance system of plasmid R1 form an operon that is autoregulated at the level of." transcription by the co-ordinated action of the Kis and Kid proteins.” Molecular microbiology vol. 5,11 (1991): 2685-93. doi:10.1111/j.1365-2958.1991.tb01977.x. Our teem hope that our killing system could help other IGEM teams in the future.

Suggestions for choosing operons

  As a target sequence bound by RNA polymerase and some transcription factors, the promoter has a regulatory and control effect on transcription initiation, determines the initiation of gene expression process and under what conditions, thereby affecting the strength of gene expression. Therefore, choosing the right promoter is important for biological research. We recommend the T7 promoter, a strong promoter from the T7 phage that can specifically react to T7RNA polymerase, and a sequence that initiates transcription of the T7 phage gene. T7RNA polymerase is highly promoter specific and only transcribes DNA downstream of the T7 promoter in T7 phages.

  Ribosomal binding sites can bind ribosomes to the translation initiation site correctly, and can also control the accuracy and efficiency of mRNA translation initiation. In prokaryotes, transcription and translation are the two main processes of gene expression. The transcription-obtained mRNA is recruited and bound to ribosomes by the 5′-end RBS sequence, and the translation process of the gene is further realized. Therefore, the strength of RBS directly affects the binding efficiency of ribosomes and determines the translation strength to a certain extent. I hope that the IGEM team that will do gene expression research in the future will not ignore RBS. We use and recommend BBa_B0034, which is medium-high in strength and has been verified many times by others.

  Terminator T is the DNA sequence that gives the RNA polymerase transcriptional termination signal. The role of terminators is strong and weak, and the selection of terminators will directly affect the amount of mRNA. In experiments, it is often necessary to replace different terminators to find the one that "fits" the gene of interest. Terminators, on the other hand, have a high failure rate of gene synthesis because of their short sequence and stem and ring structure. We recommend that future IGEM teams choose us to use a dual terminator BBa_B0015 consisting of BBa_B0010 and BBa_B0012, which works best and is used by the most people.

  Our team think those operons could be good suggestions to other IGEM teams in the future.

Suggestions for choosing chassis microorganims

  E. coli Nissle1917 has a clear genetic background, which is convenient for transgenetics

Suggestions on social investigation and statistics.

  When conducting social surveys, it is important to pay attention to the diversity of the population. Occupation, age, gender and other factors should be taken into account. At the same time, when conducting social investigations, do not only through one medium, but also investigate in many ways and find more platforms for promotion, such as WeChat public accounts, vibrato, QQ, Weibo and so on.

  When sending out questionnaires, be sure to pay attention to sensitive words, such as our program is a probiotic for relieving depression. When we investigate the situation of depressed people online, we should not use words such as "depression", because of the Internet system, the use of such sensitive words will lead to the ban of online surveys, but also cause some depressed people to have a negative attitude. You can use an abbreviation or other symbol to represent this sensitive word.

Suggestion on Education

  In terms of education, future IGEM teams should take into account the education population. It is better to be a student or an elderly person, as they are more accessible and listen to lectures. The location of education can be selected at the teacher or nursing home, which will be very convenient. That's all our team's suggestion on Education.

Suggestion on Inclusivity

  In terms of inclusion, our team recommends that future IGEM teams target not just the same group of people, but also different countries and different groups of people in the same country. So our team chose different languages and dialects from different regions to help our audience understand better. That's our suggestion on Inclusivity.

Collaborations and Alliances

  Team-to-team collaboration is very important and how to find other teams is a big problem. The advice provided by this team is to be sure to participate in meetings such as CCIC. At conferences like this, each IGEM team shares its own innovations and projects for others to consider. At the same time, if the team encounters other teams with the same or similar projects, you can form alliances with them. Our team formed an intestinal alliance with other teams related to gut probiotics. We help each other in the alliance and grow together.