Latest revision as of 14:43, 1 October 2019

NanoLuciferase reporter for BACTH assay (with two restriction enzymes around the reporter)

BACTH Principle

The Bacterial Adenylate Cyclase Two Hybrid (BACTH) principle lies on the interaction-mediated reconstitution of a signaling cascade in Escherichia Coli bacteria. The messenger molecule involved in this cascade is the cyclic adenosine monophosphate (cAMP) produced by the adenylate cyclase. The adenylate cyclase is an enzyme catalysing the cAMP production from ATP, it physiologically participates to the cellular transmission. In this system, the Bordetella pertussis adenylate cyclase, responsible agent for the pertussis, is involved. Its catalytical domain has the particularity to may be split in two distinct parts: T18 and T25 fragments, unable to work unless they reassociate. Each part of the enzyme is fused with a protein, either the bait or the prey protein. If the proteins interact, then T18 and T25 get sufficient closer and reconstitute a functional enzyme, thus allowing cAMP production. By using cya- bacteria –bacteria for whom the adenylate gene was deleted, involving an absence of this endogenous enzyme – a BACTH could be done with the creation of two fusion proteins : the first one, fused at its N or C terminal intracellular end with T18 fragment; the second one fused with T25 fragment. The interaction of these protein of interest will lead to the adenylate cyclase reconstitution, and the cAMP produced will have a messenger role. By fixing itself to the transcriptional activator CAP, cAMP form the CAP/cAMP complex, controlling the expression of a plac inducible promoter. This promoter is placed upstream the chosen reporter gene.

Sequence and features

A reporter plasmid was created to be used for BATCH assays. This reporter plasmid is composed of a cAMP inducible promoter (CAP dependant) driving the expression of a reporter gene. We used a very well characterized iGEM part : BBa_J04450 (https://parts.igem.org/Part:BBa_J04450). Characterization of this BioBrick done by our team (link) showed that the lactose promoter present in this BioBrick was not inducing gene expression of the following gene if there is no cAMP which makes this BioBrick useful for our system.

Construction

Restriction sites were added on both 5’ and 3’ ends of the Red Fluorescent Protein gene as such:

The mRFP1 was removed and replaced by the Nano Luciferase previously amplified from a commercial vector).

BglII and BamHI are still in the biobrick in order to be able to replace the RFP gene by any a RFC25 compatible gene.

Nano Luciferase was chosen for its ability to produce luminescence without the need of ATP (ATP being the substrate of Adenylate Cyclase (AC)), it must be as available as possible and so not be used by our reporter protein) and for the high level of luminescence observed for a single protein .

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 229
Illegal BamHI site found at 769
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

Our detection system is based on the use of a BACTH. The point is to allow the induction of the gene only when the two sub-parts of AC are physically close, which only occurs when the target is present in the sample. The reassembly of AC enables cAMP production, Following by the activation of a CAP dependent promoter. ⁽¹⁾. If you want to use this system, you can use an AC deficient bacteria strain (BTH101) that is not able to produce cAMP endogenously to prevent any transcription from CAP dependant promoter such as lactose promoter ⁽²⁾. For the choice of the promoter, we decide to use the lactose promoter (a CAP dependent promoter) and we have demonstrated its repression in the absence of an exogenous source of cAMP (in the AC deficient bacterial strain). ⁽³⁾.

References

⁽¹⁾ Fields S, Song O. A novel genetic system to detect protein–protein interactions. Nature [Internet]. 1989

⁽²⁾ Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. PNAS [Internet]. 1998

⁽³⁾ Karimova G, Gauliard E, Davi M, P.Ouellette S, Ladant D. Protein–Protein Interaction: Bacterial Two-Hybrid. 2017