Difference between revisions of "Part:BBa K3128004"
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K3128001 short</partinfo>
+
<partinfo>BBa_K3128004 short</partinfo>
  
 
===BACTH Principle===
 
===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.  
+
The Bacterial Adenylate Cyclase Two Hybrid (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.
 
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.
 
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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==Sequence and features==
 
==Sequence and features==
 
<div style="text-align:justify;">   
 
<div style="text-align:justify;">   
−
The BioBrick needed to have a cAMP inducible promoter (CAP dependant) like lactose promoter, followed by the reporter gene so it can be used as part of the BACTH. To do that we used a very well characterized iGEM part: BBa_J04450 (https://parts.igem.org/Part:BBa_J04450). Characterization of this BioBrick done by our team (link) showed that the lactose promoter present in this BioBrick doesn’t allow transcription of the following gene if there is no cAMP which makes this BioBrick useful for our system.
+
A reporter plasmid was created to be used for BATCH assays. This reporter plasmid is composed of a cAMP inducible promoter (CAP dependant) driving the expression of a reporter gene.
 +
We used a very well characterized iGEM part : BBa_J04450(https://parts.igem.org/Part:BBa_J04450). Characterization of this BioBrick done by our team (link) showed that the lactose promoter present in this BioBrick was not inducing gene expression of the following gene if there is no cAMP which makes this BioBrick useful for our system.
  
 +
=Construction=
 
Restriction sites were added on both 5’ and 3’ ends of the Red Fluorescent Protein gene as such:  
 
Restriction sites were added on both 5’ and 3’ ends of the Red Fluorescent Protein gene as such:  
  
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Then a classic cloning was done to replace the RFP gene by the Nano Luciferase gene (from Promega vector “pNL1.1[Nluc]”).  
+
The mRFP1 was removed and replaced by the Nano Luciferase previously amplified from a commercial vector).
−
Nano Luciferase was chosen for its capacity to produce luminescence without the need of ATP (ATP being the substrate of AC it must be as available as possible for the cAMP production and so not be used by our reporter protein) and for her high luminescence per protein capacity.  
+
 
 +
Nano Luciferase was chosen for its ability to produce luminescence without the need of ATP (ATP being the substrate of Adenylate Cyclase (AC)), it must be as available as possible and so not be used by our reporter protein) and for the high level of luminescence observed for a single protein .  
 
</div>
 
</div>
  
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===Usage and Biology===
 
===Usage and Biology===
 
<div style="text-align:justify;">   
 
<div style="text-align:justify;">   
−
Our detection system is based on the use of a BACTH. The point is to allow the induction of the gene only when the two sub-parts of AC are physically close, which only occurs when the target is present in the sample. The re-formation of AC then enables cAMP production, which will activate a CAP dependent promotor allowing the transcription of the following gene. <sup>(1)</sup>. For this we needed to use an AC deficient bacteria strain (BTH101) that that can’t produce endogenic cAMP to prevent any transcription from CAP dependant promoter such as lactose promoter <sup>(2)</sup>. As for the promoter, we chose to use the lactose promoter which is a CAP dependent one, and we demonstrated in our contribution that it is totally repressed in the absence of exogenous cAMP in this AC deficient bacterial strain, thus preventing preventing any transcription of the following gene : the reporter. <sup>(3)</sup>.  
+
Our detection system is based on the use of a BACTH. The point is to allow the induction of the gene only when the two sub-parts of AC are physically close, which only occurs when the target is present in the sample. The reassembly of AC enables cAMP production, Following by the activation of a CAP dependent promoter. <sup>(1)</sup>. If you want to use this system, you can use an AC deficient bacteria strain (BTH101) that is not able to produce cAMP endogenously to prevent any transcription from CAP dependant promoter such as lactose promoter <sup>(2)</sup>. For the choice of the promoter, we decide to use the lactose promoter (a CAP dependent promoter) and we have demonstrated its repression in the absence of an exogenous source of cAMP (in the AC deficient bacterial strain). <sup>(3)</sup>.  
  
  

Revision as of 17:50, 20 September 2019

NanoLuciferase reporter for BACTH assay (with two restriction enzymes around the reporter)

BACTH Principle

The Bacterial Adenylate Cyclase Two Hybrid (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.

Sequence and features

A reporter plasmid was created to be used for BATCH assays. This reporter plasmid is composed of a cAMP inducible promoter (CAP dependant) driving the expression of a reporter gene. We used a very well characterized iGEM part : BBa_J04450(https://parts.igem.org/Part:BBa_J04450). Characterization of this BioBrick done by our team (link) showed that the lactose promoter present in this BioBrick was not inducing gene expression of the following gene if there is no cAMP which makes this BioBrick useful for our system.

Construction

Restriction sites were added on both 5’ and 3’ ends of the Red Fluorescent Protein gene as such:


796px-T--Grenoble-Alpes--PLac_RFP_%2B_sites.png


The mRFP1 was removed and replaced by the Nano Luciferase previously amplified from a commercial vector).

Nano Luciferase was chosen for its ability to produce luminescence without the need of ATP (ATP being the substrate of Adenylate Cyclase (AC)), it must be as available as possible and so not be used by our reporter protein) and for the high level of luminescence observed for a single protein .


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Our detection system is based on the use of a BACTH. The point is to allow the induction of the gene only when the two sub-parts of AC are physically close, which only occurs when the target is present in the sample. The reassembly of AC enables cAMP production, Following by the activation of a CAP dependent promoter. (1). If you want to use this system, you can use an AC deficient bacteria strain (BTH101) that is not able to produce cAMP endogenously to prevent any transcription from CAP dependant promoter such as lactose promoter (2). For the choice of the promoter, we decide to use the lactose promoter (a CAP dependent promoter) and we have demonstrated its repression in the absence of an exogenous source of cAMP (in the AC deficient bacterial strain). (3).


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