Team:Tsinghua

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Team

Always a Harry potter fan. One to three

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Project

'E Col Transporter' Overview This project is destined to generate several E. Coli strains which will cooperate with each other to move forth and back and transport any desired protein along a gradient. Module composition: binding, releasing, transition, movement Binding SH3 domain has high affinity for proline-rich peptides. The small size and high affinity are ideal for our carrier design. In our experiment, we used a short peptide with a Kd of 3.67μM (Y. Jacquot, et al., 2007) as the binding motif and planned to transport protein substrates with this peptide sequence. This part comprises expression of three proteins, namely, OmpA-SH3 protein, which functions as the binding vehicle, OmpA-mCherry protein, which functions as the positive control, Proline-rich containing mCherry protein, which functions as the binding substrate. Releasing It’s difficult to release the substrate from strong binding, and hence protease is called into play. HIV-protease is readily available and its high efficiency, low molecular weight and high specificity constitutes fine candidate. We only need to add the substrate sequence between OmpA and SH3 sequence and the protease will cut off the binding cassette. In order to segregate the action, we’ll generate a single bacteria strain expressing the protease. Transition We need to shift between two states, the binding state and the releasing state. This molecular device is well-known for its lag in phase change, which is well adapted for our application. The lag during the phase transition is appropriate for movement and transport. Movement Kinase system is well utilized in E. Coli movement control. We’ll again use LVA tagged kinase to drive E. Coli towards the target.
























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Parts

We built a host of parts during our project. The idea is that after every small step, we store our sequence as a biobrick part.

This strategy marks our progress and facilitates future use of these sequences.

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Experiment

Our experiments are carefully recorded on a daily basis. Through the series of records, we can see our joys and sorrows.

Besides, we made the records for the purpose that our experiments can be repeated one day by someone else, thus contributing to the exploration of the unknown.

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Support

Our project is supported by School of Life Sciences, Department of Physics, Academy of Arts and Designs in Tsinghua.

Besides, we cited from a series of references.

The whole project is the work of Tsinghua iGEM Team comprising of 11 undergraduates. During the process, we shared the duty but every one has distinct focuses. The project was mainly designed by Yunxiao Zhang. Majority of the molecular cloning is done by Pan Deng, Guanqiao Li, Liyuan Zhu, and Xiao Shi. As for the protein purification and verification of our parts, Li Li, Yu Zhao and Chen Chen did most of the lab work. Our advisor Xiaozhi Fu kindly provided the guidance for using the equipment and chemicals in the lab while our director Dr. Guoqiang Chen helped us draft the flowchart of this project.

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Human Practice

We put safety as our first priority and made a detailed safety brochure.

We also devoted efforts to publicize synthetic biology and to cooperate with other teams. The teams in China held a summer meetup to discuss our progress and share our resources. We also held a lecture introducing iGEM and our project in Tsinghua Univeristy. To get further support, we sought the cooperation of iGEM Team at Macquire, Australia.

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