Part:BBa_K3128019

COMP fused with T18 subpart of Bordetella Pertussis AC under constitutive promoter

Sequence and features

This biobrick is composed of the external membrane Clickable Outer Membrane Protein (COMP) protein fused at its N-terminal end to a 54 aa Gly-Gly-Ser Linker (GGS) followed by the T18 sub-part of the Bordetella Pertussis adenylate cyclase (AC) .
This biobrick is under a constitutive promoter J23101, thereby COMP fused with T18 protein is constitutively expressed leading to a large amount of recombinant protein in the outer membrane at any time.

COMP correspond to OmpX-WT which has a TAG codon (Y121F' mutation) which can be used to introduce any Non Natural Amino acid outside the cell (more informations). This biobrick constitutes one of the final part of the NeuroDrop device, because the non natural amino acid allows to present an aptamer to the external environment of the bacteria, and the intracellular part constitutes the signalling cascade of the biomarker detection (BACTH assay).

This BioBrick is intended to opperate with BBa_K3128020.
These two biobricks are the negative condition of the mBACTH -free sub-parts condition-.
COMP are fused to the adenylate cyclase sub-parts at their N-terminal ends. The fusion protein move freely in the bacterial outer membrane, but they are not forced to get closer by any mean.
The reconstitution of the adenylate cyclase in this condition is only due to random occurrence between both parts.
The signal measured is considered as background noise.

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1300
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 841
  Illegal NgoMIV site found at 1251
  Illegal AgeI site found at 1057
  
• 1000
  COMPATIBLE WITH RFC[1000]

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 Outer-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