Composite

Part:BBa_K1650007

Designed by: Daniel Stukenberg   Group: iGEM15_Marburg   (2015-09-18)

Composite carrying the Lac-inducible CDI-operon, Lac-inducible RFP and LacI

Characterization

The composite part contains a lac-inducible CDI operon which gives your strain the capability to inhibit growth of bacterial cells that express BamA in their outer membrane (most gram negative bacteria). Additionally the part contains mRFP1 (BBa_E1010) driven by another lac-inducible promoter as induction control. Moreover the composite part contains LacI. IPTG-induced cells harboring BBa_K1650007 express CdiB which forms a beta-barrel structure. The CdiA protein is transported through CdiB and is displayed on the cell surface. When getting in contact with BamA of a target cell, the C-terminal domain is delivered into the target cell acting as pore forming protein thus destroying the proton motoric force of the target cell. BBa_K1650007 could be used to improve transformation efficiency. Induced CDI+ cells would kill false positive cells.

MR_pic_FAres.jpg
Effect of CDI expression on the ratio of CDI+ cells to target cells. The x-axis shows the time in coculture of CDI+ and target cells and the y-axis shows the corresponding normalized cell count. Over time the ratio for induced CDI+ cells increases and decreases for uninduced CDI+ cells. The used method was flow cytometry.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 9139
    Illegal PstI site found at 3585
    Illegal PstI site found at 3770
    Illegal PstI site found at 4265
    Illegal PstI site found at 4340
    Illegal PstI site found at 5372
    Illegal PstI site found at 5519
    Illegal PstI site found at 5658
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 9139
    Illegal NheI site found at 947
    Illegal NheI site found at 970
    Illegal NheI site found at 2218
    Illegal NheI site found at 2241
    Illegal PstI site found at 3585
    Illegal PstI site found at 3770
    Illegal PstI site found at 4265
    Illegal PstI site found at 4340
    Illegal PstI site found at 5372
    Illegal PstI site found at 5519
    Illegal PstI site found at 5658
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 9139
    Illegal BglII site found at 2148
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 9139
    Illegal PstI site found at 3585
    Illegal PstI site found at 3770
    Illegal PstI site found at 4265
    Illegal PstI site found at 4340
    Illegal PstI site found at 5372
    Illegal PstI site found at 5519
    Illegal PstI site found at 5658
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 9139
    Illegal PstI site found at 3585
    Illegal PstI site found at 3770
    Illegal PstI site found at 4265
    Illegal PstI site found at 4340
    Illegal PstI site found at 5372
    Illegal PstI site found at 5519
    Illegal PstI site found at 5658
    Illegal NgoMIV site found at 5393
    Illegal NgoMIV site found at 5888
    Illegal NgoMIV site found at 7463
    Illegal NgoMIV site found at 9017
    Illegal AgeI site found at 781
    Illegal AgeI site found at 893
    Illegal AgeI site found at 2827
    Illegal AgeI site found at 2939
    Illegal AgeI site found at 4028
    Illegal AgeI site found at 5202
    Illegal AgeI site found at 5255
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4752
    Illegal BsaI site found at 9583
    Illegal BsaI site found at 10004
    Illegal BsaI.rc site found at 4260
    Illegal BsaI.rc site found at 4527
    Illegal BsaI.rc site found at 13265


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