Composite

Part:BBa_K2082238

Designed by: Pascal Schmidt   Group: iGEM16_Bielefeld-CeBiTec   (2016-10-14)


Beta-lactamase under the control of a modified lacZ promoter

Beta-lactamase under the control of an optimized lacZ promotor with a binding site for cI of phage 434- BBa_K2082211

This part is designed for the possible transcriptional activation of the reporter gene beta-lactamase for possible selection possibilities. Several point mutations were integrated in the PlacZ seuqence to create a bit leaky promoter which is inducible by two specific designed fusion proteins. The OR1 binding site upstream of the promoter is the specific binding site for the cI repressor protein of the phage 434. The transcription can be activated by adding the two fusion proteins which are interacting with each other. The first fusion protein has to contain a DNA binding domain cI of the phage 434 fused with the target protein. A possible fusion protein would be the BioBrick BBa_K2082225. The second fusion protein has to contain an activation domain like RpoZ (omega subunit of the RNA polymerase I of E. coli) fused with a binding protein with the ability to interact with the target protein. A possible partner for the BBa_K2082225 fusion protein could be BBa_K2082221. The first fusion protein binds to the DNA. Now an interaction of the two fusion proteins results in a recruitment of the RNA polymerase at the optimized lacZ promoter. This consults in an increased activity of the promoter and a higher transcription of the reporter gene downstream of the promoter.
Figure 1: Sequence of the optimized PlacZ and the OR1 binding site. Illustrated is the -35-Region and -10-Region of the promoter. The uppercase letters demonstrate the mutated base-pairs in opposite to the normal PlacZ promoter.

This part is also included in the part BBa_K2082251 to accomplish a knock-out of the rpoZ gene in E. coli with a simultaneously knock-in of this reporter construct of the bacterial two-hybrid system.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1024
    Illegal NheI site found at 1047
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 817


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