Composite

Part:BBa_K2336037

Designed by: Ziyang Xiao   Group: iGEM17_HUST-China   (2017-10-24)

PmrA-PmrB(LBT5)-PmrC-GFP

The sensing part of our circuit. LBT will be a part of membrane protein PmrB. The lanthanon can be bound with LBT. Then PmrA will be phosphorylated and activate PmrC. In the end, GFP behind PmrC will be expressed.

Usage and biology

LBT（lanthanide binding tag） is a kind of protein which can bind with the lanthanide ions. We change the Fe3+ binding site in PmrB system with LBT5. Then the whole sensing system from Salmonella can sense the concentration of lanthanide ions nearby. When the concentration of lanthanide ions reach the threshold, the lanthanide can be bound with LBT5. Then the PmrB will activate PmrA by phosphorylating it. Phosphorylated PmrA is going to activate promoter PmrC in next step. In the end, GFP behind PmrC will be expressed. You can get the relative concentration of lanthanide according to the fluorescence intensity. Figure 1 shows the circuit of PmrA-PmrB(LBT5)-PmrC-GFP.

Figure1:PmrA-PmrB(LBT5)-PmrC-GFP

Characterization

This is a part for sensing part. LBT5 can bind the lanthanide ions and excite fluorescence by the sensing system, which can show the concentration of lanthanide. In our experiment, we let the engineering bacterium express GFP, which means that the lanthanide ions in the water can be sensed.

Improvement

We designed 12 LBTs to change the iron ion binding site, because we aim to test their binding force to select the most sensitive system. As we didn’t have enough time, we couldn’t let all the 12 LBTs be changed successfully. After competition of this year, HUST-China will get other LBTs changed to form the whole system. If we make it, we will link the sensing part and capture part to form the whole circuit which can sense the concentration of lanthanide ions and capture the lanthanide ions right after it. When our engineering bacteria finish their task, they can be recycle by silicon board.

Sequence and Features


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
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