Though Nissle 1917 doesn't have growth advantage if it's released in the environment, we still cared about it's escape. We wanted to find a way to make it survive harder if it was leaked through babies' excreta. However, since we aimed to produce a long-term treatment, it's hard to let Nissle suicide immediately in the environment while maintain surviving in babies' gut. According to this bottleneck, we had't find a good solusion for a long time.
When we met team UCAS-China at the 6th Conference of China iGEMers Community (CCiC), things had turned around. Their project builed a universal platform based on safety and efficiency considers for microbial therapies, called Ark.micro. Ark.micro consists of temprature-sensitive circuits as is shown below.
They inserted the TEV recognition sequence into the link part of transcription factors so that TEV protease can cut the transcription factors Cl434 to inactivate it. Meanwhile, they put the TEV protease coding gene under the promoter of corresponding Cl434 so that TEV and TF inhabit each other. Once there is a small disturbance, the balance will lean to one side to act. TEV's activity is triggered by coldness. When coldness comes, the TEV protease can make the target gene (GOI, goal of interest) expressed.
In our condition, the target gene is doc( a toxin protein gene). When coldness comes( at 25℃), TEV will activate doc to make our Nissle suicide. The escape frequency is low to 1/1000. Through UCAS-China's system, Nissle can kill itself when it is released in the environment. By transferring Ark.micro platform carrying toxin protein into our system, we can make Nissle precisely respond to the temperature change.
We met team Shanghai Tech in Shanghai Regional IGEMers Meetup at New York University Shanghai. Our projects both focused on putting e.coli into human intestine, so we have lots in common. After the conference, our team invited ShanghaiTech_China team to our school for further communication.
After detailed understanding their project, we proposed some ideas about the reference on how to tranform bacteria Nissle. Moreover, we provided our imagination about regulating in order to avoid enhancing competition or destroying intestinal flora.
On the other hand, they gave us some advice on how to handle a more than large loop circuit in modeling. With their inspiration, we can obtain a more meaningful result by dividing the whole model into several relatively independent parts. Furthermore, we discussed the parameter setting and accuracy in depth with them.
After the visit, we swapped more targeted advice on bacteria design and polished each other’s project.
Our team and Fudan-TSI were both set up in January this year, and we kept communicating with each other since that time. Our lab wasn't equipped with nanodrop, but it's important to our experiment. Team Fudan-TSI let us use their nanodrop from time to time. That really helped us a lot, otherwise we can't carry out our experiment smoothly.
We provided a sample of our plasmid containing the Tetracycline resistance gene to them to help them build their library. Also, they shared their guidebook with us and we gained a lot. The continuous communication between members of the two teams contributes to promoting respective projects.
We met SCUT_China at the 6th Conference of China iGEMers Community (CCiC). Our projects both focused on enhancing the acid-resistance of an e.coli strain. We had a lot in common, so we communicated deeply through the conference progress.
Our design of enhancing nissle's acid-resistance is to knock out nissle's HNS gene. This design is based on a research .
However, we didn't carry out this part smoothly. We had trouble knocking out Nissle's H-NS gene for more than a month. After our discussion with SCUT_China, we surprisingly found that this research was did by team SCUT_China's PI, so we immediately asked them details of knocking the HNS gene. They told us we could try extending homogenous arm. They also gave us the strain of E. coli MG1655 with H-NS gene knocked out developed by their laboratory for reference. Based on their advice and E.coli strain, we finally knocked out Nissle's H-NS gene sucessfully.
: Xianxing Gao, Xiaofeng yang, Jiahui Li, Yan Zhang, Ping Chen, Zhanglin Lin. Engineered global regulator H-NS improves the acid tolerance of E.coli[J]. Microb Cell Fact (2018) 17:118
In the process of our project, we once wanted to use machine learning to predict the best hybrid promoter we can choose. However, it turned out that the training set of the hybrid promoter is not large enough, so unfortunately we gave up the promoter strength prediction finally. Though it doesn't work in our system, it can be used in other team's system. Team JiangnanU_China achieved an anti-phage strategy against infection in different infection periods. Therefore, it's essential to control the promoter strength for the potential leakage or inclusion body problems. We gave them the advice that it might be interesting to use ANN for promoter strength prediction and gave them a paper about the similar prediction method to establish an ANN promoter strength prediction model for them. Though this method doesn't work out in our situation, it helps JiangnanU_China to select more suitable promoters designed by computer simulation.