Team:HZAU-China/Attributions

Attributions

Our project was designed by Zhujun Xia and all circuits were designed by Zhujun Xia, YinQing Zeng and Mo Qiqin.

Wet Lab

Gene Editing

sifA and sipD were knocked out completed by Zhujun Xia, Zhuoqi Huang and Wenxin Hu also gave assistance.

Plasmid Construction

Intracellular Environment-Dependent Circuits:

The intracellular environment-dependent circuits were designed by Mo Qiqin and Zhujun Xia.

The intracellular environment-dependent circuits were constructed by Mo Qiqin. Ruonan Tian also gave assistance.

Chemical Control System:

The ATc induction chemical control system was constructed by He Yang and Zhuoqi Huang also gave assistance.

The TetR-eGFP fusion protein expression system for TetR expression measurement was constructed by He Yang and Zhuoqi Huang also gave assistance.

Targeting Specificity and Surface-Displaying:

RGD-inserted OmpA circuit clone and construction of Salmonella SL1344_RS05255 (OmpA coding sequence) were completed by YinQing Zeng and Lingyu Zhong.

The circuit of surface-display system was constructed by Lingyu Zhong. Optimation of OmpA in E. Coli was completed by Zhujun Xia.

Plasmid Standardization:

Plasmid standardization was completed by Zhiqing Guo and Zhuoqi Huang by using 3A Assembly.

Plasmid Submission:

Submitted plasmids (Pcs2-eGFP-GSDMD FL, Pcs2-eGFP-GSDMD-N275 and two GSDMD full length mutations) were constructed by Zhiqing Guo and Wenxin Hu.

The description of submitted plasmids by ATP assay and cell phenotype via transfection was designed and completed by Zhujun Xia.

Bacterial Phenotype Verification

Bacterial phenotype verifications were designed and completed by He Yang, including the OD measurement and CFU measurement of chemical control system.

Cell Phenotype Verification

Cell phenotype verifications were designed and completed by Zhujun Xia, including the verification of chemical control system, and targeting specificity, all the related circuits and the demonstration of the final work system.

Biosafety Verification

Safety verification was designed and completed by Zhujun Xia, which demonstrated our project is safe enough to be used.

InterLab

InterLab was conducted by Xichen Rao, Zhujun Xia, Ruonan Tian and Heng Heng also gave assistance. Our data passed the check of the Measurement Committee successfully.

Dry Lab

Mathematical model and app about salmonella infection and GSDMD release were built by Songtao Chen.

ATc model which predicted the relationship between concentration of ATc and concentration of GSDMD was built by Xichen Rao.

The modeling on adjusting the strength of RBS and ATc with fixed GSDMD-N concentration was completed by Yini Miao.

The modeling of the promoter sifA and promoter ent was completed by Ruonan Tian.

Software

The applet of "Bacterial Colony Counter" was designed and programmed by Shuguang Wang.

Human Practice

Collaboration and communication with other teams were completed by Yinqing Zeng and all the other team members.

Handicraft manufacture was designed and completed by Yinqing Zeng.

Questionnaire preparation, the advertisement of synthetic biology and preparation of popular science articles were completed by Heng Heng.

Two posters for Eurasian exchange meetings and CCIC were completed by Ruonan Tian.

Website

Our wiki framework was designed and programmed by Shuguang Wang and our artist were Yuying Xiang and Kening Chen.

Our blog wass built by Shuguang Wang.

Others

The plate reader machine operation was conducted by Mo Qiqin.

Chief cleaner of the laboratory was Xichen Rao.

Acknowledgements

For each parts of experiments, our appreciations are as follow:

Cell lines

Thanks to Prof. Shan Li’s laboratory, Huazhong Agricultural University, for sharing the cell lines of HEK293T, HELA, MCF7, iBMDM, so that our experiment can process successfully.

Thanks to Dr. Feng Shao’s laboratory, for sharing the cell line of GSDMD-/- HELA cells, which provides us great support.

Thanks to Dr. Yi Zeng and Dr. Ms Hong Fan, Shanghai Institute of Biochemistry and Cell Biology, CAS, for sharing the cell line of MDA-MB-231.

Bacteria

Thanks to Prof. Shan Li’s laboratory, Huazhong Agricultural University, for sharing the strain of Salmonella enterica var. Typhimurium SL1344.

Thanks to Dr. Chenli Liu from SIAT CSynBER for generously sharing the material (Strain: E.coli K-12 MG1655 with mCherry in the genome).

Gene Knock In and Knock Out

Thanks to Prof. Shan Li’s laboratory, Huazhong Agricultural University, for providing the protocol and material of gene editing based on two-step allelic exchange, which provides us with great help.

Thanks to Pan Chu, team leader of iGEM-2016, Huazhong Agricultural University, for teaching us multigene editing based on CRISPR and λ-RED.

Plasmid Backbone

Thanks to Prof. Shan Li’s laboratory, Huazhong Agricultural University, for sharing the plasmid backbone (Pcs2-eGFP) to us.