Team:Shanghai HS/Design

Solve the harm caused by cyanobacteria

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Megamenu

Cyanobarrier: Solve the harm caused by cyanobacteria

We anticipate our solution to aid in protecting the environment and avoiding people from getting sick because of drinking contaminated water.

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Design

• Obtain gene of MlrA

  MlrA is a name given to the enzyme that breakdown microcystin-LR. We initiated our design by searching for alternative MlrA gene sequences in the National Center for biotechnology information. Cluster analysis was performed by the software MAGE to compare those genes. We were eventually able to select the optimal genes of MlrA from two species Novosphingobium sp. THN1 and Sphingopyxis sp. ACM-3962. We named them as MlrA2 and MlrA3 respectively during our study.

• MlrA expression

  Our method is inserting an external MlrA gene into the competent cell(E.coli) to express it. At first, we constructed a recombinant plasmid with MlrA. Then increased the yield by cultivating them in the petri dish and extract the plasmid we want. To obtain mlrA in a pure form, what we did was to disassemble the grown competent cell and protein analysis. French pressure cell press was employed for this purpose. We separated the pressed cell by centrifugation to get the pure MlrA protein. In order to predict the properties of the MlrA gene, TMHMM2.0
  (http://www.cbs.dtu.dk/services/TMHMM/), a technique that provides predictions of transmembrane helices in proteins was utilized, but it could only accommodate numerical values and estimate that there will be several transmembrane domains. We used a more direct program known as Raptor X
  (http://raptorx.uchicago.edu/StructurePropertyPred/predict/) where it reveals the specific structure of a MlrA protein using the protein sequence. This showcased that the MlrA protein has a 7 a-Helix, a distinct feature of a membrane protein. At the beginning of our study, we expected that the MlrA was a soluble protein, but later found out that the protein was expressed as precipitate which means it is a membrane protein.

• Examine the Microcystin degradation

  HPLC separate solvents according to their polarity. We mixed the microcystin-LR with the MlrA at different proportion and left them to react for a specific amount of time, then injected the reacted mixture to the HPLC device. After tuning the machine to the right way and programming the progress.we could see the amount of micocystin-LR degraded to find out how efficient does the MlrA work.

• Immobilizing the MlrA

  To not waste any of MlrA we made with painstaking effort, immobilizing it and reusing it is the best way to reduce cost of production. We plan to use alginate beads to seal the MlrA. The MlrA is mixed with the sodium alginate solution, and dropped to the calcium chloride solution. When the two solution meet, they instantly react to form jelly. Thus, MlrA can be hold tight in the beads after solidification. We can fill a column by those beads and when the water with microcystin-LR passes through the column, it can be purified.

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