Difference between revisions of "Part:BBa K2888008"
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| − | We attempt to link the MC-degrading enzyme and the cyanobacteria phage-originated lysozyme to produce a protein that possesses the function of both enzymes by genetically modifying E.coli . We also make a solution with the Bugbuster and the putative functional enzymes to perform cyanobacteria lysis and MC degradation simultaneously. Furthermore, we purposely add 6x | + | We attempt to link the MC-degrading enzyme and the cyanobacteria phage-originated lysozyme to produce a protein that possesses the function of both enzymes by genetically modifying E.coli . We also make a solution with the Bugbuster and the putative functional enzymes to perform cyanobacteria lysis and MC degradation simultaneously. Furthermore, we purposely add 6x His tag into our sequence in order to improve the efficacy and efficiency of expression and purification; it will also strengthen effects of binding to Ni-NTA column, making it possible to immobilize proteins and lyse cyanobacteria repeatedly in our prototype device. |
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Latest revision as of 00:09, 18 October 2018
fused chimerical gene with 6x His tag (lysozyme and mlrA)
We attempt to link the MC-degrading enzyme and the cyanobacteria phage-originated lysozyme to produce a protein that possesses the function of both enzymes by genetically modifying E.coli . We also make a solution with the Bugbuster and the putative functional enzymes to perform cyanobacteria lysis and MC degradation simultaneously. Furthermore, we purposely add 6x His tag into our sequence in order to improve the efficacy and efficiency of expression and purification; it will also strengthen effects of binding to Ni-NTA column, making it possible to immobilize proteins and lyse cyanobacteria repeatedly in our prototype device.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1349
Illegal AgeI site found at 884 - 1000COMPATIBLE WITH RFC[1000]
Introduction
We have designed, based on our original goal, a basic part of chimeric lysozyme and mlrA gene with a linker sequence in between in order to create an enzyme that retain the both the function of lysing the cyanobacteria and denaturing their toxin MCLR. This part is intended to resolve the ultimate problem of cyanobacteria pollution. Therefore, the composite part contains our promoter, RBS, 6✖️His tag, lysozyme gene, a linker in between, mlrA gene and a terminator. During some of the experimental trials, we also managed to add a Sumo tag before the 6✖️His tag in order to increase the solubility of the enzyme.
Experience
Through Nested PCR, we are able to successfully infuse the lysozyme- mlrA gene into the PSB1C3 backbone. The linearized plasmid after the infusion is seen below on lane 2 and 8 of the gel. However, due to limited time, we were only able to focus on the function of lysozyme, which is part BBa_K2888002 and part BBa_K2888003.
Reference
1. Mehta, K. K., Evitt, N. H. & Swartz, J. R. Chemical lysis of cyanobacteria. Journal of Biological Engineering 9, (2015).
2. Chen, J. et al. Degradation of Microcystin-LR and RR by a Stenotrophomonas sp. Strain EMS Isolated from Lake Taihu, China. International Journal of Molecular Sciences 11, 896–911 (2010).
3. Shimizu, K. et al. How microcystin-degrading bacteria express microcystin degradation activity. Lakes & Reservoirs: Research & Management 16, 169–178 (2011).
4. Khan, F., He, M. & Taussig, M. J. Double-Hexahistidine Tag with High-Affinity Binding for Protein Immobilization, Purification, and Detection on Ni−Nitrilotriacetic Acid Surfaces. Analytical Chemistry 78, 3072–3079 (2006).