Composite

Part:BBa_K2389070

Designed by: Rochelin Dalangin   Group: iGEM17_UAlberta   (2017-10-26)

GFP reporter under lac promoter + lacI

This construct codes for the green fluorescent protein (GFP) under the control of the lac promoter, which has the catabolite activator protein (CAP) binding site, and the lacI gene under constitutive expression. This reporter is a modified version of BBa_K611016, where we replaced the arabinose-induced promoter of the lacI gene with a constitutive promoter (BBa_J23106). As a result, induction with arabinose is no longer necessary for lacI expression, and we also believe that this constitutive promoter will lead to more consistent expression levels of lacI. For our project, The titratable expression of GFP serves as an indirect reporter for the strength of protein-protein interactions in the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system.

Briefly, the BACTH system is a method for indirectly measuring protein-protein interaction strength (Karimova et al.; Battesti and Bouveret). It relies on the reconstitution of the two catalytic halves of adenylate cyclase (T18 and T25) for cAMP production and regulation of reporter genes. The commonly used endogenous lac operon can be used as a reporter, but we found that the b-galactosidase assay was fairly time consuming and required the use of harmful chemicals. We designed this fluorescent reporter to serve as a secondary means of characterizing our BACTH (pT8-O) constructs (BBa_K2389010, BBa_K2389020, BBa_K2389030, BBa_K2389040) and to provide an alternative method for screening for high affinity interactions. The presence of the CAP binding site allows for the upregulation of GFP expression with increased levels of cAMP. Further fine tuning of the reporter is regulated by the binding of the lacI repressor to the operator site of the promoter.

References: Battesti, Aurélia, and Emmanuelle Bouveret. "The Bacterial Two-Hybrid System Based on Adenylate Cyclase Reconstitution in Escherichia Coli." Methods 58.4 (2012): 325-34. Print. Karimova, Gouzel, et al. "A Bacterial Two-Hybrid System Based on a Reconstituted Signal Transduction Pathway." Proceedings of the National Academy of Sciences 95.10 (1998): 5752-56. Print.
Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1098
    Illegal NheI site found at 1121
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2299
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 870


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