Composite

Part:BBa_K2888008

Designed by: SBS_SH_112144   Group: iGEM18_SBS_SH_112144   (2018-10-07)
Revision as of 23:26, 17 October 2018 by Yinchizhou18 (Talk | contribs)


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 histag 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1349
    Illegal AgeI site found at 884
  • 1000
    COMPATIBLE 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.

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