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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.
Protocols
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. Three kinds of protocols: Protocol 1 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 microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences). Protocol 2 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). After 3 hours’ culture at 30 °C, each culture (200 μL) was induced for 7 hours with inducers of different concentrations. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences). Protocol 3 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). After 6 hours’ culture at 30 °C, each culture (200 μL) was centrifuged at 4000 r.p.m. for 10 minutes and was suspended in 200 μL of fresh LB medium containing inducers of different concentrations for 4 hours. Then the fluorescence intensity of cultures was measured by microplate reader (Thermo) or LSRFortessa flow cytometer (BD Biosciences).
To see more our protocols for test and analysis, please click here.
Result
The aromatic compounds concentration was parallelly measured three times by microplate reader. The induction ratios are as follow. According to the table, the water samples didn’t induce the manipulated bacteria obviously and each kind of aromatic compounds was below the detection limit. According to the experiment, Weiming Lake’s water sample didn’t induce manipulated bacteria obviously. It can be drawn that benzoic acid, aminosalicylic acid and phenylog derivatives concentration of Weiming Lake are all under the water pollution standard of our country (GB8978-1996). The conclusion are drawn as follow, water from Weiming Lake is not natural water, but running water from taps, which was purified by complex procedures. It is anticipatable that the water from Weiming Lake is up to standard and there are no aromatic compounds of concentrations over the detection limits (around 1 μM) within.
