Team:LN-Shiyan-China/Parts
Parts
Our Submission Parts:
BBa_K1667005
This part includes two individual DNA domains. Constitutive promoter tunes the expression of downstream opdA gene with further help from RNA thermometer. RNA thermometer provides a temperature sensitive post-transcriptional regulation on opdA gene, which initiate the opdA translation around 32°C. OmpA signal peptide guides the secretion of opdA protein to the outside of host strain. Then opdA enzyme specifically degrades organophosphorus pesticide appeared, through hydrolysis. Upon UV light, RecA（SOS）promoter drives the transcription of downstream ccdB suicide gene, whose protein expression interferes DNA sysnthesis and lead to cell dealth. In this way, we can wipe out our genetic engineered bacteria by giving UV lights under manual control to avoid secondary pollution. Without UV light, RecA promoter won’t be activated, so the normal growth and activity of genetic engineering bacteria will be preserved well to release functional opdA protein under temperature control.
Experimentally Verification Scheme:
Bacteria strain BL21 (DE3) of colibacillus genetically engineered bacteria which contains our target plasmid goes through extended culture in our LB liquid substrate for sixteen hours under the temperature of 30°C, then centrifuge the culture for five minutes with the speed of 5000rpm under the temperature of 4°C and use sterile MSM (mineral base medium) to wash and sediment the bacteria, then use fresh MSM culture substrate to dilute the bacteria. Inoculate the bacteria to MSM culture substrate which contains 10μg/mL chlorpyrifos or methyl-parathion (the final concentration of bacteria is 106 CFU/mL), and culture it with the speed of 160rpm under the temperature of 37°C while shaking it. Regularly get samples from the solution and measure the concentration of organophosphorus pesticides in the solution. Measure the OD600 of the culture to indicate the growth of the bacteria, use the culture solution which is not inoculated as a blank control while using the colibacillus inoculated with the bacteria strain BL21 (DE3) which does not contain target plasmid as a reference control. Adopt gas chromatography to measure the concentration of organophosphorus pesticides in the culture.
Extraction of organophosphorus pesticides: Get 5mL MSM solution which is inoculated and cultured as above, add 25mL acetonitrile into it and shake it violently for five minutes, then add 2.5g sodium chloride into it and shake it violently, place the solution quietly for 30 minutes then laminarize it, get 1mL organic phase in the upper layer and dry it with pressure blowing concentrator, add 1mL acetone into it to dissolve above dry organic phase again.
Gas chromatography detecting conditions: Use Shimadzu 2014C gas chromatographic analyzer, equip it with fire photometric detector (FPD) and quartz capillary chromatographic column (length of column is 30 meters, inner diameter of column is 0.53mm, thickness of membrane layer is 1μm, RESTEX, USA), flow speed of nitrogen is lmL/min while the flow speeds of air and hydrogen are respectively 81.8mL/min and 3.2mL/min. The temperatures of vaporizing chamber, column and detector are respectively 250°C, 150°C and 250°C. The initial temperature of column is 150°C and should be kept for three minutes. Then the temperature of column should be heated to 250°C using the speed of 8°C/min and the final temperature should be kept for eight minutes. The quantity of input sample is 10μL.
Later, different change influences culture temperatures, PH values and inoculation quantity have on degrading organophosphorus can be set. Among these conditions, the organophosphorus pesticides are not degraded at the temperature below 32°C while they begin to be degraded at the temperature of over 32°C. The degradation effects generally must be good at neutral PH values. Generally, the more the inoculation quantity is, the better the degrading effects are.
