Difference between revisions of "Part:BBa K3128018:Experience"

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. You might know this bacterium; it is the responsible agent for the pertussis disease. It adenylates cyclase catalytic domain has the particularity to be splittable in two distinct parts: T18 and T25 sub-parts, unable to work 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 promoter lactose by initiating transcription of the following gene.
This promoter is placed upstream the chosen reporter gene.

[http://2015.igem.org/Team:TU_Eindhoven Eindhoven-2015] iGEM project’s aim was to develop a “universal membrane sensor platform for biosensors”.
This year, Team Grenoble-Alpes is designing a new tears biosensor system based on [http://2015.igem.org/Team:TU_Eindhoven Eindhoven-2015]’s project. Both projects have a common base, the same receptors are used at the external surface of bacteria : Clickable Outer Membrane Protein X(COMP).

OmpX is an outer membrane protein with the C- and N-termini in the intracellular domain. To be able to use OmpX as a scaffold, a unnatural amino acid needs to be introduced. This can be done by implementing the amber stop codon TAG in one of the loops of OmpX via a mutation. With a specific tRNA an azide-functionalized amino acid can be built in, which can be used for the SPAAC click chemistry reaction using DIBO functionalized groups, this modified protein is called COMP. The complex aptamer fixed to a COMP is then named a COMB for Clickable Outer Membrane Biosensor.

The Grenoble-Alpes team aims to develop an Outer membrane Bacterial Adenylate Cyclase Two Hybrid (mBACTH).
In this case, the two adenylate cyclase sub-parts are fused to the N-terminal ends of COMPs with a Gly-Gly-Ser Linker (GGS) of 54 amino acids - in order to ensure a sufficient flexibility -.
When COMBs catch the extracellular target, they get closer, thus allowing the reconstitution of a functional adenylate cyclase due to the physical proximity of the two sub-parts.
The enzyme is operational again and produce a high quantity of cAMP (around 17,000 mmol of cAMP formed per mg of adenylate cyclase per minute), the molecule responsible for the signal transduction in the bacteria.

cAMP molecules diffuse to the cytoplasm of the bacterium and interact with catabolite activator proteins (CAP) in a ratio 1 to 1. Yhen two cAMP-CAP complexes are needed to activate the expression of the gene under the control of the lactose promoter.
Because of the high quantity of cAMP diffusing in the cytoplasm of the bacterium (2), the reporter gene is continously activated as long as cAMP is produced.

The high enzymatic activity (1) of Bordetella pertussis Adenylate Cyclase generates a high production of cAMP in presence of ATP in the bacterium thus activating the signalling cascade with the CAP-cAMP dependant promoter.
Hence this system is promising because it might have a great sensitivity and may drive a great signal amplification for a low amount of melocules to be detected.