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Team:Nanjing-China/deg
From 2013.igem.org
Revision as of 03:25, 25 September 2013 by GaoJian NJU (Talk | contribs)
- Degrading Enzyme
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Atrazine is quite hard to degrade naturally in the soil, which makes it persist for a long time in the environment once used. This phenomenon can be considerably severe in that atrazine would cause metabolic disorders both in animals and humankind. For many years, we didn't have an efficient solution to this problem.
However, the discovery of the super power of Arthrobacter aurescens provides us with a light of hope. This amazing species is capable of utilizing atrazine as a sole source of carbon with the help of a series of degrading enzymes which can metabolize atrazine into a kind of nontoxic substance. In our project, we utilize the most useful degrading enzyme TrzN to degrade atrazine. We even mutated the TrzN to make it degrade faster. At the same time, we found a transmembrane transporter TRM, which involves in transporting atrazine from the outside to the inside of the bacteria so that atrazine can be better degraded.
Experiments and Results
We took 12 15mL-tubes, divided them into 4 groups: Group 0; Group Wild Type; Group TrzN- and Group TrzN+, 3 tubes each. With the same protocol used to test the function of TRM, we found that TrzN could distinctively degrade atrazine with the bacteria cultivated 24 hours in 37℃ (Fig. 3-6-1). The difference between wild type and TrzN+ was distinctive, suggesting that TrzN is a degrading enzyme of atrazine.
Fig. 3-6-1 TrzN can distinctively degrade atrazine. This figure shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Group 0 means no bacteria in the cultures; cultures in Group Wild Type contained the wild type K12 bacteria; cultures in Group Vector contained the strain K12 with pGFP; and cultures in Group TrzN+ contained the bacteria which are able to express TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can conclude that TrzNis an efficient degrading enzyme in solving the problem of TrzN.
We also combined the TRM and TrzN together can make the degradation more efficient (more information about TRM. This time, we combined the TRM and TrzN together, under the same method of testing the function of TRM and TrzN, we found that the combination of TRM and TrzN could decrease the atrazine concentration in the bacteria culture to a greater extent (Fig. 3-6-2).
Fig. 3-6-2 Combining TRM and TrzN together can make the degradation more efficient. shows the atrazine concentration in supernatant of bacterial cultures, which were cultured for 24 hours in 37℃. Apart from groups shown above, cultures in Group TRM+TrzN+ contained the bacteria which are able to express TRM as well as TrzN. "*" means the difference between two groups which are connected by half square brackets is distinctive. From this chart we can see that the atrazine concentration in supernatant of Group TRM+TrzN+ was even distinctively less than that in supernatant of TRM+. This means combining TRM and TrzN is indeed a wise choice to polish up our system.
According to these data, we find that TRM can indeed transport atrazine from the outside to the inside of the bacteria, and of course, TrzN can degrade atrazine well and fast. Therefore, we could be confident of the fact that we may one day utilize our system to solve the problem of atrazine in real life.
