Composite

Part:BBa_K3654018

Designed by: Raymond Kim   Group: iGEM20_KSA_KOREA   (2020-10-27)
Revision as of 01:41, 28 October 2020 by Gomsemarii (Talk | contribs)


terminator/PlacI-RBS-mprA-T7 terminator - PT7-LacO-RBS-CouP-T7 terminator

This composite part is composed of a combination of two composite parts (BBa_K3654013 and BBa_K3654016). BBa_K3654016 is responsible for inducing the continuous expression of mprA and IPTG induction of CouP. In addition, BBa_K3654013 provides a site for regulating the expression of mScarlet.


Usage and Biology

1. Design

Our biosensor is composed of two main parts. The first part expresses mprA, which functions as a negative regulator, and the other part regulates the expression of the reporter gene. The mprA protein expressed by the constitutive promoter LacI binds to the emrR promoter and inhibits mScarlet expression. When Biosensor E. coli uptakes phenolic compounds (Fig. 1B), the mprA protein and the phenolic compound are bound, while the mprA protein is no longer bound to the emrR promoter. As a result, mScarlet becomes expressed.

800px-Biosensor_result_fig1.png

2nd Iteration As the overexpression of CouP could affect the expression of the Reporter gene, we constructed a new plasmid DNA in which LacI promoter controls the CouP expression,. We followed the same procedure as the previous experiments, and observed brighter red fluorescence when CouP was present. After digitizing the pixel intensity of the fluorescence with the help of ImageJ, we found that the fluorescence was about 20% higher when CouP was present.

Biosensor_result_fig4.png Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 1378
    Illegal PstI site found at 2350
    Illegal PstI site found at 2788
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 2350
    Illegal PstI site found at 2788
    Illegal NotI site found at 130
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2933
    Illegal XhoI site found at 159
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1378
    Illegal PstI site found at 2350
    Illegal PstI site found at 2788
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1378
    Illegal PstI site found at 2350
    Illegal PstI site found at 2788
    Illegal NgoMIV site found at 1229
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 650
    Illegal SapI.rc site found at 2691


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