Difference between revisions of "Part:BBa K3128020"

 
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<partinfo>BBa_K3128020 short</partinfo>
 
<partinfo>BBa_K3128020 short</partinfo>
  
This biobrick is composed of the external membrane OMPx muted 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 constitutive promoter allowing the bacteria to have a huge quantity of proteins located at the membrane before the target is added to the external environment (thus, before the detection).
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==Sequence and features==
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<div style="text-align:justify;"> 
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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 comes from the intermediate biobrick <partinfo>BBa_K3128016</partinfo>, for which the plac promoter has been deleted to replace it with a constitutive promoter.
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Additionally, it 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).
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</div>
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<partinfo>BBa_K3128019 SequenceAndFeatures</partinfo>
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==Usage and Biology==
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<div style="text-align:justify;"> 
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In 1989, Fields and Song demonstrated a new genetic system allowing protein-protein interaction detection <sup>(1)</sup>. 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 <sup>(2)</sup>. Nowadays, this biological technique is mostly used to show and characterize the physical interaction between two cytosolic proteins or internal membrane proteins in vivo <sup>(3)</sup>.
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===BACTH Principle===
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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.
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https://static.igem.org/mediawiki/parts/0/0b/BACTH_constructions.gif
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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.
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 +
 
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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.
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 +
===NeuroDrop Project - Membrane BACTH ===
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iGEM Grenoble-Alpes team have chose to create a membrane bacterial two hybrid by using the OmpX protein, fused with adenylate cyclase subparts.
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 +
As part of the iGEM Grenoble-Alpes 2019 project, the constitutive promoter allows the bacteria to have a huge quantity of proteins located at the membrane before the detection of the target in the external environment. 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.
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This biobrick constitutes a part of the mBACTH system that the team used as a negative control of this assay. Indeed, this biobrick with <partinfo>BBa_K3128019</partinfo> allow to have OmpX proteins fused to the two subunits of the adenylate cyclase from Bordetella Pertussis that move freely in the membrane. This condition enables the team to estimate the number of false positive in their system.
  
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https://static.igem.org/mediawiki/parts/e/ec/BACTH_1.gif
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https://static.igem.org/mediawiki/parts/f/fd/BACTH_2.gif
  
<!-- Add more about the biology of this part here
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</div>
===Usage and Biology===
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==References==
<span class='h3bb'>Sequence and Features</span>
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<sup>(1)</sup> Fields S, Song O. A novel genetic system to detect protein–protein interactions. Nature [Internet]. 1989
<partinfo>BBa_K3128020 SequenceAndFeatures</partinfo>
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<sup>(2)</sup> Karimova G, Pidoux J, Ullmann A, Ladant D. A bacterial two-hybrid system based on a reconstituted signal transduction pathway. PNAS [Internet]. 1998
  
<!-- Uncomment this to enable Functional Parameter display
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<sup>(3)</sup> Karimova G, Gauliard E, Davi M, P.Ouellette S, Ladant D. Protein–Protein Interaction: Bacterial Two-Hybrid. 2017
===Functional Parameters===
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<partinfo>BBa_K3128020 parameters</partinfo>
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Revision as of 17:58, 30 September 2019

COMP fused with T25 subpart of Bordetella Pertussis AC under constitutive promoter

Sequence and features

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 comes from the intermediate biobrick BBa_K3128016, for which the plac promoter has been deleted to replace it with a constitutive promoter.

Additionally, it 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).



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 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.

BACTH_constructions.gif

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.

As part of the iGEM Grenoble-Alpes 2019 project, the constitutive promoter allows the bacteria to have a huge quantity of proteins located at the membrane before the detection of the target in the external environment. 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.

This biobrick constitutes a part of the mBACTH system that the team used as a negative control of this assay. Indeed, this biobrick with BBa_K3128019 allow to have OmpX proteins fused to the two subunits of the adenylate cyclase from Bordetella Pertussis that move freely in the membrane. This condition enables the team to estimate the number of false positive in their system.

BACTH_1.gif BACTH_2.gif

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