Difference between revisions of "Part:BBa K3128001"

A reporter biobrick was constructed to be used with a BATCH assays. This reporter plasmid is composed of a cAMP dependant promoter (CAP dependant) driving the expression of a reporter gene.
A very well characterized iGEM part was used : BBa_J04450.
Characterization of this BioBrick done by Team Grenoble-Alpes 2019showed that the lactose promoter present in this BioBrick, which makes this BioBrick useful for our system.

Construction

BglII and BamHI restriction restriction sites were added on both 5’ and 3’ ends of the Red Fluorescent Protein gene as follow:

The mRFP1 was removed and replaced by the NanoLuciferase gene previously amplified from a commercial vector.

Nano Luciferase was chosen for its ability to produce luminescence in absence 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 function 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.

When 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.

NeuroDrop 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, followed by the transcription of the reporter that is under a CAP-cAMP dependent promoter.
To use this system, an AC deficient bacteria strain (BTH101) that is not able to produce endogenous cAMP is needed to prevent any transcription from CAP dependant promoter in abscence of interaction between both sub-parts.
For the promoter, we decided to use the lactose promoter (a CAP dependent promoter), we have demonstrated its repression in the absence of an exogenous source of cAMP (in the AC deficient bacterial strain).