Part:BBa_K3128023

OmpX-WT with Leucine Zipper and T25 subpart of Bordetella Pertussis AC under lactose promoter

Sequence and features

This biobrick is composed of the external membrane Outer Membrane Protein X Wild Type (OmpX-WT) protein fused at its N-terminal end with a 54 aa Gly-Gly-Ser Linker (GGS) followed by the T25 sub-part of the Bordetella Pertussis adenylate cyclase (AC) .
Leucine-zipper sequence is added between the signal peptide of OmpX -to express the recombiant protein in the external membrane- and OmpX gene in order to force the physical closeness of Rocombinants proteins.
Leucine zippers are peptides which contain a hydrophobic leucine residue at every seventh position.
They dimerize through interactions between their helices.
This is a strategy to emulate the target recognition by the aptamers located at the bacterial cell surface. Hence the adenylate cyclase activity will be restored through the interaction of both subparts and will induce the cAMP dependant signalling cascade.

iGEM Grenoble-Alpes designed this biobrick with the pKT25 plasmid present in the Euromedex BACTH kit (more informations in the design page).
It is an intermediate because the team want to switch the promoter lactose already present in pKT25 by a constitutive promoter.
It will allows the bacteria to have a huge quantity of proteins located in the membrane before the target is added to the external environment thus incresing the sensitivity of the NeuroDrop system.
In addition, we chose to have only 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.

This BioBrick intended to operate with BBa_K3128022.
These two biobricks are the positive condition of the mBACTH -Leucine Zipper condition-.
OmpX proteins are fused to the adenylate cyclase sub-parts at their N-terminal and to Leucine Zipper at their C-teminal
Thereby they are forced to get closer to form dimers in bacterial external membrane. The reconstitution of the adenylate cyclase in this condition forced by Leucine Zipper to simulate the precense of the target.
The signal measured is considered as the positive control of the system.

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1764
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 344
  

Usage and Biology

In 1989, Fields and Song demonstrated a new genetic system allowing the detection of protein-protein interaction ⁽¹⁾. At first, it was performed 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⁾.

Bacterial Adenylate Cyclase Two-Hybrid (BACTH)

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 the 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 from the chosen reporter gene.

NeuroDrop Project - inter-Membrane BACTH (mBACTH)

[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 called 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 with DBCO functionalized groups, this modified protein is called COMP. The complex aptamer fixed to a COMP is then called a COMB for Clickable Outer Membrane Biosensor.

The Grenoble-Alpes team aims to develop an intermembrane 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 COMPs and the aptamers 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 can produce the production of a high quantity of cAMP (around 17,000 mmol of cAMP formed per mg of adenylate cyclase per minute), the molecule responsible of the signal transduction in the bacterium.

cAMP molecules diffuse to the cytoplasm of the bacterium and interact with catabolite activator proteins (CAP). One cAMP molecule binds to one transcriptional activator CAP; then 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 continually activated as long as cAMP is produced.

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

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