Composite

Part:BBa_K2082239

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


BBa_K2082231 expanded by a second cI binding site OR2

Optimized PlacZ with double cI binding site OR1 and OR2 combined with the fusion protein SH2:cI(434)

This part is an expanded version of the BioBrick BBa_K2082231. The difference between these two parts is the new designed binding site upstream of the reporter. This part contains upstream of the OR1 binding site a second binding site OR2 for the cI(434) repressor protein. It was expected that this second binding site could consults in a stronger binding of the cI repressor protein and therefore, a better functionality 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 860
    Illegal NheI site found at 883
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 675
    Illegal AgeI site found at 787
  • 1000
    COMPATIBLE WITH RFC[1000]

Characterization

The binding strength of the repressor protein cI at our modified binding site was tested by an electrophoretic mobility shift assay (EMSA). The band shift of the doubled binding site was compared with the band shift of the single binding site construct. Different protein concentrations of the SH2:cI(434) fusion protein were added to the DNA fragments to analyze when the band shift is visible. A band shift with lower protein concentration would confirm a stronger interaction of the protein with the DNA. The agarose gel revealed no significant differences between the appearance of the band shift between the simple or the doubled binding site for cI. Therefore, the double binding site does not result in a stronger interaction of the repressor protein with the DNA.
Figure 1: EMSA results.The DNA fragments containing the single OR1 and the double OR1 and OR2 binding site were applied. The SH2-cI protein was added with an amount of 0, 0.5, 5 and 50 pmol. A band shift only appear with a protein concentration of 50 pmol for both binding site variations.

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