Composite

Part:BBa_K2336037:Design

Designed by: Ziyang Xiao   Group: iGEM17_HUST-China   (2017-10-24)
Revision as of 19:38, 31 October 2017 by IreneLi (Talk | contribs)

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PmrA-PmrB(LBT5)-PmrC-GFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2116
    Illegal XhoI site found at 2155
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1704
    Illegal AgeI site found at 2881
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2804
    Illegal SapI.rc site found at 691

Design Note

We made full use of the two-component system found in Salmonella. We try to change the Iron binding motif to 12 lathanide binding tags in the membrane protein PmrB. Then PmrB can activate PmrA by phosphorylating it. Phosphorylated PmrA is going to activate promoter PmrC and GFP can be expressed. In the end, the sensing system can detect the concentration of lanthanide nearby.


Resources

Our LBT.PmrA-PmrB(Fe).PmrC-GFP were synthesized by IDT. Then we used SOE (splicing by over-lapping extension) to change the Iron binding motif to 12 lathanide binding tags in the membrane protein PmrB. Moreover, we use restriction enzyme and ligase to fuse the PmrA-PmrB(LBT) and PmrC-GFP.

References

Engineering Bacterial Two-Component System PmrA/PmrB to Sense Lanthanide Ions