Difference between revisions of "Part:BBa K3128016"

This biobrick is composed of the external membrane OMPx WT protein fused at its N-terminal end with a 54 aa GGS linker (BBa_K3128010) followed by the T25 subunit of the Bordetella Pertussis adenylate cyclase. This biobrick is under plac promoter, thus the expression of the OmpX protein can be activated with IPTG.

This biobrick has a TAG codon (Y121F' mutation) which can be used to introduce any Non Natural Amino acid outside the cell (more informations).

iGEM Grenoble-Alpes designed this biobrick thanks to the pKNT25 plasmid contained in the Euromedex BACTH kit (more informations in the design page). It is an intermediate because the team want to change the plac promoter with a constitutive promoter. It allows the bacteria to have a huge quantity of proteins located in the membrane before the target is added to the external environment (thus, before the detection). In addition, the team needs just one gene under cAMP inducible CAP dependant plac promoter in its final system: the reporter gene, in order to do a membrane Bacterial Adenylate Cyclase Two Hybrid.

In 1989, Fields and Song demonstrated a new genetic system allowing protein-protein interaction detection ⁽¹⁾. At first, it was done in Saccharomyces cerevisiae yeast and it was called the yeast two-hybrid assay (Y2H). In 1998, Ladant and al. described the system in bacteria ⁽²⁾. Nowadays, this biological technique is mostly used to show and characterize the physical interaction between two cytosolic proteins or internal membrane proteins in vivo ⁽³⁾.

BACTH Principle

The 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.

NeuroDrop Project - Membrane BACTH

iGEM Grenoble-Alpes team have chose to create a membrane bacterial two hybrid by using the OmpX protein, fused with adenylate cyclase subparts.