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Team:Nanjing-China/deg
From 2013.igem.org
Revision as of 06:35, 27 October 2013 by GaoJian NJU (Talk | contribs)
- Degradation
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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 (more information about TRM).
Experiments and Results
Firstly, we took 12 15mL-tubes, divided them into 4 groups: Group 0; Group Wild Type; Group Vector and Group TrzN+, 3 tubes each. Then, corresponding materials were added into each group. After 24h of incubation in 37℃ (Fig. 3-6-1), the atrazine concentration in solution of Group TrzN+ was remarkably less than that in solution of other groups.
Fig. 3-6-1 TrzN can distinctively degrade atrazine. This picture 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 TrzN is an efficient degrading enzyme in solving the problem of TrzN.
Combining TRM and TrzN together can make the degradation more efficient. 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 This picture 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.
