Team:OUC-China/Proof Of Concept
Overview
Our project was inspired by the overuse of the antibiotics. As we all know, antibiotics play an important role in human health and agricultural production. However, at the same time, the overuse of it is causing the great development of antibiotic resistance genes and serious environmental pollution. Thus we hope our genetic circuits which is based on the whole-cell biosensors could test the antibiotics in the environment, especially the water environment, quicker and more convenient.
1. Cells can survive in the different water environment
Since we hope our biosensors could be used to detect the antibiotics in the water environment,such as seawater, aquaculture tail water and wastewater. We designed a lot of experiments to ensure that our cells can survive in the terrible environment. Therefore, we performed a plate-reader experiment, where cells were grown in various media: LB culture, mixes of LB and wastewater, wastewater and Ultrapure water. We measured the fluorescence intensity and optical density (OD) every hour. The results confirm our hypothesis that E. coli can survive in the different environment, as fluorescence and OD600 increases in all samples compared with negative control.
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Figure 1
Fig 1. OD600 and relative fluorescence intensity of cells cultured in the different media.We set the group of cells with pUC19 as negative control and the cells with T7(tetO)-sfGFP as positive control. (a)-(b) OD600 and relative fluorescence intensity of cells cultured in the sewage inlet (c)-(d) OD600 and relative fluorescence intensity of cells cultured in the aquaculture tail water (e)-(f) OD600 and relative fluorescence intensity of cells cultured in the seawater
The results confirm our hypothesis that E. coli can survive in the different environment, as fluorescence intensity and OD600 both are higher in most of samples compared with negative control. However, we found that the cell density of our cells cultured in the aquaculture water is far less than the positive controls. But our cells can keep well cell density in a short time in the aquaculture water.
2. Detect
To achieve the goal of antibiotics detection, we designed genetic circuits consisted of a coding sequence of allosteric Transcription Factors (tetR, ctcS and mphR) and 3WJdB or sfGFP genes under the control of T7 promoters. We linked antibiotic resistance genes into the Basic circuit by molecular cloning. TetM was assembled downstream of ctcS or tetR and controlled by the same constitutive promoter (Pc2). Successful colonies were verified by colony PCR then confirmed with sequencing.
When antibiotics are absent, aTFs bind to the inducible promoter and prevent RNA polymerase from initiating transcription, thus repressing the expression of reporter gene. If antibiotics are present, the repressors will be no longer binds to the operator, thus the intensity of fluorescence will increase.
Since we wanted to ensure that our biosensors could work as our expectation, we performed several plate reader experiments, where cells transformed with the biosensor were grown in media with different concentrations of antibiotics.
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Figure 2
a) the fluorescence of tetR-tetM- T7(tetO)-3WJdB incubated in different concentrations of tetracycline.
b) the fluorescence of ctcS-tetM- T7(tetO)-sfGFP incubated in different concentrations of chlortetracycline.
From the results, we could see that our engineered cells can produce fluorescent signal when the antibiotics (in this two cases, the antibiotics are tet and CTE) reaches a certain concentration.What’s more, with the increase of the antibiotic’s concentrations, max relative fluorescence intensity increases accordingly.
It should be mentioned that all of our experiments followed the iGEM Safety Committee rules and policies ,there is no leakage of our engineered bacteria.