Difference between revisions of "Part:BBa K3225014"

Latest revision as of 18:05, 25 October 2020

J23101-LuxR-pLux-GFP

This device is designed to detect N-(3-oxohexanoyl)-L-homoserine lactone (OHHL quorum sensing signal molecular of E.carotovora). It consists of LuxR(quorum sensing transcriptional factor, BBa_C0062) drove by a constitutive promoter J23101 and GFP reporter drove by the binding promoter of LuxR(pLux, BBa_R0062).

Sequence and Features

Results

1.Results in vivo

From figure1, we found that the GFP intensity increased after OHHL incubation, even 1nM OHHL, which means LuxR could bind to OHHL efficiently. It showed that LuxR - pLux could be a highly sensitive detector of OHHL.

Figure 1.The fluorescence intensity over the time with different concentration of OHHL treatment. The inducer OHHL was added to the culture after 60 min incubation.

2.Results in vitro(cell-free system).

After the cellular sensing characterization, we tested the binding efficiency of LuxR to OHHL in cell-free systems(Fig. 3). After the overnight cell-free incubation, it showed that the circuit of LuxR-GFP was responsible to AHL with the detection limit of 1 nM.

Figure 2. The cell-free fluorescence output induced by a series of AHL concentrations. FLU was measured after overnight incubation

Improvements

Team: iBowu-China 2020 Time:2020/10/26

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 reporter.

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).

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.