Part:BBa_K3296004
mlrA2
This is the core part of our experiment. It is responsible for the degradation of microcyctin. It can help us to make the microcyctin antitoxic. This part is derived from a specie called Novosphingobium sp. THN1 based on a paper in China. And we also modified the codon inside the gene in order to make it fit the vector (BL21DE3, DH5α) we employed and there for we the protein will be better expressed. MlrA2 is from Novosphingobium sp. THN1(NCBI: AEC46646.1).
Usage and Biology
BBa_K3296004 mlrA2
Methods:
1. replicating the MlrA2 DNA through a technology called PCR, Gel Electrophoresis and Extraction of MlrA2
2. combining the gene MlrA2 with plasmids, and transformed the plasmids to DH5-alpha, a strain of E. coli.
3. Senting the extracted plasmid to Sangon, a biotech company, for DNA sequencing after a series of procedures.
4. transforming the plasmid to E. coli BL21(DE3), another type of E-coli characterized in producing protein, to express the targeted protein.
In order to determine whether the protein we finally purified is MlrA, we take 12 different samples from different periods of the experiment and let these samples run SDS-Page gel.
Figure 7A The result of the SDS-Page gel electrophoresis
Line 1: MlrA2 total sample
Line 2: MlrA2 supernatant sample
Line 3: MlrA2 precipitate sample
Line 4: MlrA2 precipitate sample after low speed centrifugation
Line 5: MlrA2 supernatant sample before high speed centrifugation
Line 6: MlrA2 supernatant sample after high speed centrifugation
Line 7: MlrA2 precipitate sample after membrane lysed
Line 8: MlrA2 supernatant sample after membrane lysed
Figure 7C The result of the SDS-Page gel electrophoresis
Line 17: MlrA2 supernatant sample after membrane lysed (same as sample 8)
Line 18: Impure MlrA2 sample that first flow through the nickel column
Line 19: The sample after 5mM imidazole wash
Line 20: The sample after 10mM imidazole wash
Line 21: The sample after 20mM imidazole wash
Line 22: The sample after 200mM imidazole wash
The result of the SDS-Page gel electrophoresis is unexpected. We can only observe very small amounts of protein at the target strip of the elution samples. However, the supernatant solution and flow through do exist higher concentration of protein at the upper position than the target strip. We theoretically speculate that this higher concentration of protein may be the mlrA without the SUMO tag; but it could also be just a kind of impure protein. Because of this uncertainty, we decide to try it use both the supernatant solution and the flow through to react with microcystin to verify that if these two samples contain the MlrA we really need.
By adding the protein that binds to the Ni21-NTA to the affinity column, we were able to collect the liquid that went through. We ran the SDS-Page Gel with thirteen chosen samples to verify the results of the protein purification. It turns out, we successfully purified MlrA2, indiciating that it can be devoted to degrading MC-LR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 250
Illegal AgeI site found at 379 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 136
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