Team:XMU-China/Contribution
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For the Contribution, we completed the experimental characterization of the previous parts (BBa_K2332002 and BBa_K2332004) and added the data of them to the corresponding BioBricks.
| BioBricks | Codes in the lab | Quantitative Characterization |
|---|---|---|
| BBa_K2332002 | pBLind | fluorescence / OD600 |
| BBa_K2332004 | EL222 | fluorescence / OD600 |
All of these may be helpful to other teams. We hope it will make some contribution to the iGEM community.
EL222 is from the marine bacterium Erythrobacter litoralis HTCC2594. It was first registered in 2017 and used as a natural photosensitive DNA-binding protein. And pBLind is a promoter activated by EL222 (BBa_K2332004) upon blue-light exposure (465nm). It was also registered in 2017.
We constructed “J23106-EL222-pBLind-sfgfp-pSB1c3”(Fig.1A)in E.coli BL21(DE3)to characterize its function.
Fig. 1. The illustration and characterization of the system. (A)Gene circuit illustration for the system.(B) RFUsfGFP/OD600 of the system in dark and blue-light condition was calculated as time progressed.
The system (Fig.1A) can be characterized by measuring the fluorescence intensity (λex=488 nm, λem=530 nm) and OD600 under the strictly shadowed condition and the blue-light irradiation condition. The relative fluorescent unit (RFU) normalized to OD600, RFUsfGFP/OD600, was calculated to represent the expression level of sfGFP in an average cell.
The result showed that the system maintained a well blue-light sensitivity and a higher expression level of sfGFP in the system was observed as time progressed (Fig. 1B). It demonstrated that the EL222 was able to work as a photosensitive DNA-binding protein and pBLind could be activated by EL222.
1.Jayaraman, P.; Devarajan, K.; Chua, T. K.; Zhang, H.; Gunawan, E.; Poh, C. L., Blue light-mediated transcriptional activation and repression of gene expression in bacteria. Nucleic Acids Res 2016, 44 (14), 6994-7005.