Difference between revisions of "Part:BBa K3128022"

 
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<partinfo>BBa_K3128022 short</partinfo>
 
<partinfo>BBa_K3128022 short</partinfo>
  
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 T18 subunit of the Bordetella Pertussis adenylate cyclase. This biobrick is under plac promoter in order to be activated when the cAMP messenger is present in the system.
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==Sequence and features==
Leucine zipper are known to interact by forming homodimers. This biobrick is a part of a positive control for the BACTH assay.
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<div style="text-align:justify;"> 
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This biobrick is composed of the external membrane Outer Membrane Protein x Wild Type <i>(OmpX-WT)</i> protein fused at its N-terminal end with a 54 aa GGS linker (BBa_K3128010) followed by the T18 subunit of the <i>Bordetella Pertussis</i> adenylate cyclase <i>(AC)</i>.<br>
 +
This biobrick is under promoter lactose <i>(PLac)</i>, thereby the expression of the OmpX-WT fused with T18 protein can be activated and auto-amplidied with IPTG.
 +
 
 +
iGEM Grenoble-Alpes designed this biobrick with the pUT18 plasmid present in the Euromedex BACTH kit (more informations in the design page).<br>
 +
It is an '''intermediate''' because the team want to switch the PLac promoter by a constitutive promoter.<br>
 +
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.<br>
 +
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.<br>
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<br>This BioBrick is meant to work with [https://parts.igem.org/wiki/index.php?title=Part:BBa_K3128023 BBa_K3128023].<br>
 +
These two biobricks constitute the positive condition of the '''mBACTH''' <i>-Leucine Zipper condition-</i>.<br>
 +
OmpX proteins are fused to the adenylate cyclase sub-parts at their N-terminal ends, but they are not forced to get closer and move freely in the bacterial external membrane.
 +
The reconstitution of the adenylate cyclase in this condition is only due to random occurrence between both parts.<br>
 +
The signal measured can be considered as the positive control of the system.
 +
</div>
  
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
  
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K3128022 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3128022 SequenceAndFeatures</partinfo>
  
  
<!-- Uncomment this to enable Functional Parameter display
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===Usage and Biology===
===Functional Parameters===
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<div style="text-align:justify;"> 
<partinfo>BBa_K3128022 parameters</partinfo>
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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===
 +
 
 +
https://static.igem.org/mediawiki/parts/0/0b/BACTH_constructions.gif
 +
 
 +
The BACTH principle relies 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.
 +
 
 +
https://static.igem.org/mediawiki/parts/e/ec/BACTH_1.gif
 +
https://static.igem.org/mediawiki/parts/f/fd/BACTH_2.gif
 +
 
 +
</div>
 +
 
 +
==References==
 +
<sup>(1)</sup> Fields S, Song O. A novel genetic system to detect protein–protein interactions. Nature [Internet]. 1989
 +
 
 +
<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
 +
 
 +
<sup>(3)</sup> Karimova G, Gauliard E, Davi M, P.Ouellette S, Ladant D. Protein–Protein Interaction: Bacterial Two-Hybrid. 2017

Revision as of 16:11, 8 October 2019

OmpX-WT with Leucine Zipper and T18 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 GGS linker (BBa_K3128010) followed by the T18 subunit of the Bordetella Pertussis adenylate cyclase (AC).
This biobrick is under promoter lactose (PLac), thereby the expression of the OmpX-WT fused with T18 protein can be activated and auto-amplidied with IPTG.

iGEM Grenoble-Alpes designed this biobrick with the pUT18 plasmid present in the Euromedex BACTH kit (more informations in the design page).
It is an intermediate because the team want to switch the PLac promoter 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 is meant to work with BBa_K3128023.
These two biobricks constitute the positive condition of the mBACTH -Leucine Zipper condition-.
OmpX proteins are fused to the adenylate cyclase sub-parts at their N-terminal ends, but they are not forced to get closer and move freely in the bacterial external membrane. The reconstitution of the adenylate cyclase in this condition is only due to random occurrence between both parts.
The signal measured can be 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 1632
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1173
    Illegal NgoMIV site found at 1583
    Illegal AgeI site found at 1389
  • 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 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

BACTH_constructions.gif

The BACTH principle relies 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.

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