Difference between revisions of "Part:BBa K3128001"

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).

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 .

The principle lies on the interaction-mediated reconstitution of a signalling cascade in Escherichia coli. The messenger molecule involved in this cascade is the cyclic adenosine monophosphate (cAMP) produced by the adenylate cyclase. Adenylate cyclase is an enzyme catalysing the cAMP production from ATP. It physiologically participates to the cellular transmission.

This system involves the Bordetella pertussis adenylate cyclase which is the responsible agent for the pertussis disease. Adenylate cyclase catalytic domain has the particularity to be splittable in two distinct parts: T18 and T25 sub-parts, unable to fonction unless they reassociate. Each sub-part of the enzyme is fused with a protein of interest, either the bait or the prey protein chose beforehand by the experimentator.

If two proteins interact, then T18 and T25 are bring together and reconstitute a functional adenylate cyclase enzyme thus enabling cAMP production. Using cya- bacteria – strain for whom the adenylate gene is 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 the T18 sub-part; the second one fused with the T25 sub-part.
The interaction of these proteins of interest will lead to the adenylate cyclase reconstitution, thus initiating cAMP production. The cAMP produced will act as a messenger by fixing itself to the transcriptional activator CAP, cAMP form the CAP-cAMP complex, controlling the expression of the lactose promoter by initiating transcription of the following gene.
This promoter is placed upstream the chosen reporter gene.

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⁾.