Team:OUC-China/Contribution

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• Overview
• New Parts
• New Documentation To Existing Part
• Document troubleshooting
• Reference

Overview

For the Contribution(Bronze Criterion #4), we have done a lot of works this year.
1. We added 4 new basic parts to the iGEM Registry Part page.
2. We completed the experimental characterization of the previous parts(BBa_K3189004,BBa_K864680 ). We added the data of them to the corresponding BioBricks.
3. We summarized the problems and solutions we came across while constructing our parts and reporter gene expression.
We hope it can help future iGEM teams succeed in their projects.

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New Parts

This year OUC-China 2021 first used four new basic parts and designed five new composite parts. We add all of them to the iGEM Registry. It is worth mentioning that we first used three-way junction dimeric Broccoli, 3WJdB, as our reporter gene! We hope our genetic circuits with reportable output based entirely on transcription can provide an alternative strategy for future iGEM teams. 

Name	Description	Detailed description
BBa_K3777000	3WJdB	three-way junction dimeric Broccoli, (3WJdB),a reporter gene
BBa_K3777001	ctcs	A model aTF that binds the operator (ctco) sequence in the absence of ligands Chlortetracycline
BBa_K3777003	KB2	a kleptamer RNA (KB2) which can disrupt the folding of 3WJdB
BBa_K3777011	mphA	MphA increase the intracellular concentration of EryA by phosphorylation of the drug

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New Documentation To Existing Part

This year we first used 3WJdB as our reporter gene, we completed the experimental characterization of the previous parts(BBa_K3189004,BBa_K864680). We also added their data to the corresponding BioBricks.

1.tetR(Part:BBa_K3189004)
TetR, a model aTF, can bind the operator (teto) sequence in the absence of ligands tetracycline. It was registered in 2019. We used both 3WJdB and sfGFP as reporter genes under the control of promoter T7 promoter and Ptet. We construct the circuit:tetR-T7(tetO)-3WJdB,tetR-Ptet-tetO-3WJdB in E. coli BL21(DE3) to characterize its function. That is to say, we added new documentation to tetR. We also hope our genetic circuits with reportable output based entirely on transcription can provide an alternative strategy for future iGEM teams. 

(A)

(B)

Fig 1. The fluorescence data of using 3WJdB as reporter gene when the aTF is tetR
(A)fluorescence expressed under the control of T7 promoter. (B)fluorescence expressed under the control of Ptet.

2.tetM(Part:BBa_K864680)
tetM, a tetracycline resistance gene, encodes a ribosomal protection protein. It was registered in 2012. We construct the circuit: tetR-T7(tetO)-tetM-3WJdB, tetR-T7(tetO)-tetM-sfGFP, ctcs-T7(ctcO)-tetM-sfGFP, ctcs-T7(ctcO)-tetM-3WJdB in E. coli BL21(DE3) to characterize its function. tetM gene has been used in multiple applications, including construction of engineered genetic circuits, environmental detection of antibiotics ，and so on. We hope it can increase the sensitivity of our genetic circuits.
(A)

(B)

Fig 2. The fluorescence data of two biosensors came from our laboratory experiments
(A)fluorescence data of genetic circuits tetR-tetM-T7(tetO)-3WJdB.
(B)fluorescence data of genetic circuits ctcs-tetM-T7(ctcO)-3WJdB.

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Document troubleshooting

This year we want to explore the trend of fluorescence intensity over time, so we should measure the fluorescence intensity and cell viability uninterruptedly. We first cultured our cells in the Microplate-reader and measured the data at the set time. However, we found that the cell viability is unsatisfactory. After asking our advisor and PI for help, we hypothesized that it is caused by the false method of cell culture. We designed experiments to explore the difference of three cultivation methods: 2ml ep tubes, 96-well plates with and Erlenmeyer flasks. Setting the cells cultured in Erlenmeyer flasks as the positive control, we measured the OD600 and fluorescence intensity of cells in different groups every hour.

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(B)

Fig3. (A)OD600 which characterized cell viability we measured with three different methods.
(B)fluorescence intensity we measured with three different methods. From the results we measured, we can see that the OD600 and fluorescence intensity of cells cultured in Erlenmeyer flasks is best. And the group of cells cultured in the 96-well plate produced slightly less fluorescent protein than the positive control group. Then we choose to culture our cells in the 96-well cells, incubate the cells shaking in the Micro-plate Incubator.

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Reference

[1]Corwin A. Miller, Joanne M. Ho, Sydney E. Parks, and Matthew R. Bennett,Macrolide Biosensor Optimization through Cellular Substrate Sequestration ACS Synthetic Biology 2021 10 (2), 258-264
[2]Jaeyoung K. Jung  , Khalid K. Alam  , Matthew S. Verosloff, Daiana A. Capdevila  Morgane Desmau , Phillip R. Clauer, Jeong Wook Lee, Peter Q. Nguyen , Pablo A. Pastén  ,Sandrine J. Matiasek  , Cell-free biosensors for rapid detection of water Contaminants Nature Biotechnology 2020.12,1451-1459