Team:iBowu-China/Contribution
Contribution
Except the LacZ or GFP based reporting system (Part:BBa_K3225014), bio-reporters were designed for quorum sensing molecules AHLs sensing on Lux system with different features in vivo or in vitro[1][2][3]. The reporters included fluorescent, colorimetric, and bioluminescent reporters.
The reporters were tested under quorum-sensing systems in vivo with reporters of mCherry, mScarlet-I, GFP, deGFP, LacZ, and NanoLuc. Among the characterized reporters, GFP and mScarlet-I presented similar dose-response curve and LOD[2]. With the increase of time, induction fold of the two reporters improved. For LacZ and NanoLuc reporters, their LOD were evidently lower than that of the florescent reporters (3−4 orders of magnitude lower), also they showed a faster response rate (30 min for LacZ and NanoLuc and 60 min for GFP and mScarlet-I). Among all the reporters, NanoLuc provided the lowest LOD which was 3.81*10-4 nM 3OC6HSL. Notably the decrease of incubation fold after 240 min was observed for NanoLuc (table 1).
For reporters acting in vitro, for all the reporters, the incubation folds are positively associated with the amounts of quorum sensing molecule under the quorum-sensing molecule sensor. GFP and deGFP exhibits a shorter response time that mCherry and mScarlet-I reporters. Moreover, the green fluorescent reporters possess a higher sensitivity than red fluorescent molecules. Notably, under LuxR sensor, the fluorescence output of deGFP is higher than that of GFP. Surprisingly, the green fluorescent reporters and NanoLuc showed the best LOD of lower than 4.0 nM 3OC6HSL, which is 5-10 times lower than other reporters (table 2).
Table 1: The in vivo detection limit of reporters in different incubation time.
For reporters acting in vitro, for all the reporters, the incubation folds are positively associated with the amounts of quorum sensing molecule under the quorum-sensing molecule sensor. GFP and deGFP exhibits a shorter response time that mCherry and mScarlet-I reporters. Moreover, the green fluorescent reporters possess a higher sensitivity than red fluorescent molecules. Notably, under LuxR sensor, the fluorescence output of deGFP is higher than that of GFP. Surprisingly, the green fluorescent reporters and NanoLuc showed the best LOD of lower than 4.0 nM 3OC6HSL, which is 5-10 times lower than other reporters (table 2).
Table 2: The in vitro detection limit of reporters in different incubation time.
References:
1. Chugani, S., & Greenberg, E. P. (2010). LuxR homolog-independent gene regulation by acyl-homoserine lactones in Pseudomonas aeruginosa. Proceedings of the National Academy of Sciences of the United States of America, 107(23), 10673–10678.
https://doi.org/10.1073/pnas.1005909107
2. Anal. Chem. 2019, 91, 23, 15284–15292, Publication Date:November 5, 2019
https://doi.org/10.1021/acs.analchem.9b04444
3. Peng Zhang, Huibao Feng, Junzhu Yang, Hao Jiang, Hongjun Zhou, Yuan Lu,
Detection of inorganic ions and organic molecules with cell-free biosensing systems,
Journal of Biotechnology,Volume 300,2019,Pages 78-86,ISSN 0168-1656,
https://doi.org/10.1016/j.jbiotec.2019.05.011.