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Team:Peking/HumanPractice/ModeliGEM
From 2013.igem.org
Practical Analysis
In order to test whether our toolkit works practically, we took water samples from our beloved Weiming Lake. Is she pure as she appears? Or is she polluted by aromatic compounds? Weiming Lake is a famous scenic spot in Peking University. In Chinese, "Weiming" means "too beautiful to be named". Many people in history failed to name it so a famous scholar gave the lake this name.
Located in the center of the campus, the lake was once a royal garden in Qing Dynasty. Today it is still regarded as a sacred lake by many visitors. On the hill of the southern part to Weiming Lake, there is a pagoda named Boya. It is an imitation of ancient tower. The combination of the lake and the tower forms a distinctive landscape. Students in Peking University can be seen reading and studying beside the lake. Thus，testing water sample from this lake is meaningful for us.
Water from Weiming Lake in Peking University was sampled at 15:30 and 19:30, sterilized by 0.22 um filter. LB solution was prepared by using each of the water samples. Antibiotics were added into two tubes containing the two water samples to prepare 50 ml engineered bacteria medium. After 12 hours cultivation according to the inducing process, OD600 and fluorescence515 of the samples were tested. Then, aromatic compounds were calculated by dose-response curves.
Methods
Strains and growth media. E.coli Top10 was used for all the experiments and cultured in Luria–Bertani (LB) medium. Kanamycin (10 μg/mL), ampicillin (50 μg/mL) and chloramphenicol (170μg/mL) were added as appropriate. Protocol E.coli was grown in LB medium overnight at 37 °C and then diluted 100-fold in fresh LB medium in 96-well plates (Corning Incorporated, 3599). Then each culture (200 μL) was induced for 12 hours at 30°C with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by a microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).
Result
The concentration of aromatic compounds was parallelly measured three times with a microplate reader and the induction ratios are showed below. The result shows that the concentration of benzoates, salicylic acids, and biphenyl derivatives to which biosensor XylS, NahR and HbpR response respectively is lower than the detecting limit of our biosensors. The Weiming lake is free from these kinds of aromatic pollution!
