Part:BBa_K3296005
MlrA3
This is the core part of our experiment. It is responsible for the degradation of microcyctin. Through the reaction with microcyctin it will linearlize it and make no toxic. We developed this gene based on BBa_K2888001, which is designed by team SBS_SH_112144. Since last team has failed in expressing the protein, we decided to design a new one in order to get the protein expressed. Thus, we looked up NCBI and found two species that contain genes similar to the one precious team had used. MlrA2 preset in a species called Sphingomonas sp. For this part, we optimized the codon in order to have a better expression result: the codon within MlrA2 is preferred by the plasmid vector, E.coli by which our gene will be well expressed. MlrA3 is from Sphingopyxis sp. ACM-3962.
Usage and Biology
BBa_K3296005 MlrA3
Methods:
1. replicating the MlrA3 DNA through a technology called PCR, Gel Electrophoresis and Extraction of MlrA3
2. combining the gene MlrA3 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 7B The result of the SDS-Page gel electrophoresis
Line 9: MlrA3 total sample
Line 10: MlrA3 supernatant sample
Line 11: MlrA3 precipitate sample
Line 12: MlrA3 precipitate sample after low speed centrifugation
Line 13: MlrA3 supernatant sample before high speed centrifugation
Line 14: MlrA3 supernatant sample after high speed centrifugation
Line 15: MlrA3 precipitate sample after membrane lysed
Line 16: MlrA3 supernatant sample after membrane lysed
Figure 7D The result of the SDS-Page gel electrophoresis
Line 23: MlrA3 supernatant sample after membrane lysed (same as sample 16)
Line 24: Impure MlrA3 sample that first flw through the nickel column
Line 25: The sample after 5mM imidazole wash
Line 26: The sample after 10mM imidazole wash
Line 27: The sample after 20mM imidazole wash
Line 28: 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 MlrA3, 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 244
Illegal AgeI site found at 373 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 130
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