Difference between revisions of "Part:BBa K3128004:Experience"

(→‎Applications of BBa_K3128004)
 
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Complete article : (link)
 
Complete article : (link)
  
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With a Bacterial Adenylate Cyclase Two Hybrid (BACTH) system the rapporteur gene have to be overexpressed when the two sub-parts of Adenylate Cyclase (AC) T18 and T25 are physically close thus enabling cAMP production. Those two parts are going to get closer and stick together only when the target is present.
+
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.
−
The produced cAMP will then activate CAP dependent promoter allowing the transcription of the following gene.
+
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.
−
As shown in our contribution (link), the lactose promoter which is a CAP dependant promoter act as a repressive promoter when there is no cAMP thus preventing any transcription of the following gene : the rapporteur gene.
+
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 cAMP (in the AC deficient bacterial strain), thus preventing preventing any transcription of the following gene : the reporter (see table and figure 1).
−
AC deficient bacteria (BTH101) that can’t produce endogenic cAMP are use in this system to prevent any false positive results.
+
To resume, the gene has to be expressed/overexpressed only when cAMP is produced and there needs to have a clear difference when the sub-parts are brought together by the target or when the target is not here and the sub-parts remain free.
−
To resume, the gene have to be expressed/overexpressed only when cAMP is produced and there need to have a clear difference when the sub-parts are bring together by the target or when the target is not here and the sub-parts are free.
+
  
−
To prove that the rapporteur gene work in our system different conditions were tested.
 
  
−
First the leak of our rapporteur when there is no cAMP was measured by only transforming the plasmid containing the BioBrick PLac_NanoLuc in BTH101.
+
To prove that the reporter gene efficiently works in our system different conditions were tested.
 +
First the leak of our reporter when there is no cAMP was measured by transforming the plasmid containing the BioBrick PLac_NanoLuc in BTH101.
 +
 
 
https://2019.igem.org/wiki/images/thumb/7/70/T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png/595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png
 
https://2019.igem.org/wiki/images/thumb/7/70/T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png/595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png
  
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Then the “free sub-parts” condition was tested by transforming two plasmids in BTH101: pUT18 containing the AC sub-part T18 and pKT25_NLuc containing both the AC sub-part T25 and the BioBrick PLac_NanoLuc. In this condition T18 and T25 don’t stick together, nevertheless they can randomly come close to each other and produce cAMP. This is the baseline measurement of our system.
+
Then the free sub-parts condition was tested by co-transforming two plasmids in BTH101: pUT18 containing the AC sub-part T18 and pKT25_NLuc containing both the AC sub-part T25 and the BioBrick PLac_NanoLuc.
 
https://2019.igem.org/wiki/images/thumb/b/b5/T--Grenoble-Alpes--Plasmid_pUT18_pKT25_NLuc.png/800px-T--Grenoble-Alpes--Plasmid_pUT18_pKT25_NLuc.png
 
https://2019.igem.org/wiki/images/thumb/b/b5/T--Grenoble-Alpes--Plasmid_pUT18_pKT25_NLuc.png/800px-T--Grenoble-Alpes--Plasmid_pUT18_pKT25_NLuc.png
  
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At last the “target is here” condition was tested, to simulate the presence of the target and the physical connexion between both sub-parts Leucine-Zipper (LZ) were used. LZ have the capacity to form homodimer and so were added at the end of both sub-parts making them able to stick to each other thus restoring the AC activity. Two plasmids were transformed in BTH101: pUT18-LZ containing the AC sub-part T18 in fusion with a LZ and pKT25-LZ_NLuc containing both the AC sub-part T25 in fusion with a LZ and the BioBrick PLac_NanoLuc.
+
At last the target detection condition was tested.
 +
Leucine-Zipper (LZ) were used to simulate the presence of the target and the physical connexion between both sub-parts. LZ have the capacity to form homodimer and so were added at the end of both sub-parts making them able to stick to each other thus restoring the AC activity. Two plasmids were co-transformed in BTH101: pUT18-LZ containing the AC sub-part T18 fused with a LZ and pKT25-LZ_NLuc containing both the AC sub-part T25 fused with a LZ and the BioBrick PLac_NanoLuc.
 +
 
 
https://2019.igem.org/wiki/images/thumb/6/6b/T--Grenoble-Alpes--Plasmid_pUT18LZ_pKT25LZ_NLuc.png/800px-T--Grenoble-Alpes--Plasmid_pUT18LZ_pKT25LZ_NLuc.png
 
https://2019.igem.org/wiki/images/thumb/6/6b/T--Grenoble-Alpes--Plasmid_pUT18LZ_pKT25LZ_NLuc.png/800px-T--Grenoble-Alpes--Plasmid_pUT18LZ_pKT25LZ_NLuc.png
  
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If there is a notable difference of luminescence between the “free sub-parts” and the “target is here” then it will mean that our rapporteur gene is working in our system.
+
If there is a notable difference of luminescence between the free sub-parts and the target detection then it will mean that our reporter gene could work in our system. It will also show that the on/off switch of the transcription depending on cAMP is working.
  
−
For the assay :
+
=The assay=
 
Bacterial culture were induced with 0.5mM of IPTG at Optic Density 0.6.
 
Bacterial culture were induced with 0.5mM of IPTG at Optic Density 0.6.
−
The subtract for Nano Luciferase (furimazine) was added as follow, for 50uL of bacterial culture in a well, 49uL of NanoGlo Assay Buffer and 1uL of NanoGlo Assay Substrate were added then wait 5 minutes before the measurement.
+
The subtract for Nano Luciferase (furimazine) was added as follow : for 50uL of bacterial culture in a well, 49uL of NanoGlo Assay Buffer and 1uL of NanoGlo Assay Substrat were added.
−
All the measures have been measured in Relative Luminescence Units (RLU) in a plate NUNC 96 wells. Two different bacterial culture (sample) were measured each time in duplicate (not for the 24 hours condition).  
+
All the measures are expressed in Relative Luminescence Units (RLU) in a NUNC 96 wells plate. Two different bacterial cultures (sample) were assessed each time in duplicate (except for the 24 hours condition). Blank was done with non-transformed BTH101 (RLU = 300) and subtracted from the measurements.
−
Blank was done with BTH101 non-transformed (RLU = 300) and subtracted from the measurements.
+
  
−
Results :
+
=Results=
  
−
https://2019.igem.org/wiki/images/thumb/1/16/T--Grenoble-Alpes--NLuc_Table_1.png/800px-T--Grenoble-Alpes--NLuc_Table_1.png
+
https://2019.igem.org/wiki/images/e/e7/T--Grenoble-Alpes--NLuc_Fig_1.png
  
  
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https://2019.igem.org/wiki/images/thumb/5/58/T--Grenoble-Alpes--NLuc_Table_2%27.png/800px-T--Grenoble-Alpes--NLuc_Table_2%27.png
+
https://2019.igem.org/wiki/images/4/4f/T--Grenoble-Alpes--NLuc_Fig_2.png
  
  
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https://2019.igem.org/wiki/images/thumb/7/76/T--Grenoble-Alpes--NLuc_Table_3.png/799px-T--Grenoble-Alpes--NLuc_Table_3.png
+
https://2019.igem.org/wiki/images/a/a3/T--Grenoble-Alpes--NLuc_Fig_3.png
  
−
 
+
=Conclusion=
−
 
+
−
Conclusion :
+
  
 
https://2019.igem.org/wiki/images/thumb/8/87/T--Grenoble-Alpes--NLuc_Graph_Conclu.png/800px-T--Grenoble-Alpes--NLuc_Graph_Conclu.png
 
https://2019.igem.org/wiki/images/thumb/8/87/T--Grenoble-Alpes--NLuc_Graph_Conclu.png/800px-T--Grenoble-Alpes--NLuc_Graph_Conclu.png
 +
 
Measurement of the Nano Luciferase assays of the 3 conditions.
 
Measurement of the Nano Luciferase assays of the 3 conditions.
  
−
There is a significant luminescence intensity difference between the assay with or without LZ which means that when the target is present and bring the two parts together the reporter gene (here Nano Luciferase) is overexpressed compared to the “free-parts” assay. With this reporter BioBrick it is possible to tell the difference when the two sub-parts are close and when they are free and so tell if the theorical target is present or not.
+
Those measurements highlight two major things with our reporter Biobrick:
−
In realistic condition the difference will not be as flagrant as here because the LZ system is more efficient to bring the parts together than the assay done with the target and aptamer (see the full system).
+
First, there is a significant luminescence gap between the assay without endogenous adenylate cyclase (grey) and with the free sub-parts (orange) which means that there is a significant leak of our reporter protein due to random occurrences between both T18 and T25.
 +
However here is a significant difference between the assay with or without LZ which means that when the target is present and brings the two parts together the gene (here Nano Luciferase) is overexpressed. With this BioBrick it is then possible to see the difference between both condition: if the two sub-parts are close and if they are free and so tell if the theoretical target is present or not.
 +
In realist conditions the difference will probably not be as flagrant as here because the LZ system is more efficient to bring the parts together than the assay done with the target and aptamer (see the full system).
 +
 
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 14:44, 1 October 2019


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K3128004

Complete article : (link)

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. 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. 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 cAMP (in the AC deficient bacterial strain), thus preventing preventing any transcription of the following gene : the reporter (see table and figure 1). To resume, the gene has to be expressed/overexpressed only when cAMP is produced and there needs to have a clear difference when the sub-parts are brought together by the target or when the target is not here and the sub-parts remain free.


To prove that the reporter gene efficiently works in our system different conditions were tested. First the leak of our reporter when there is no cAMP was measured by transforming the plasmid containing the BioBrick PLac_NanoLuc in BTH101.

595px-T--Grenoble-Alpes--Plasmid_PLacNanoLuc.png

Then the free sub-parts condition was tested by co-transforming two plasmids in BTH101: pUT18 containing the AC sub-part T18 and pKT25_NLuc containing both the AC sub-part T25 and the BioBrick PLac_NanoLuc. 800px-T--Grenoble-Alpes--Plasmid_pUT18_pKT25_NLuc.png

At last the target detection condition was tested. Leucine-Zipper (LZ) were used to simulate the presence of the target and the physical connexion between both sub-parts. LZ have the capacity to form homodimer and so were added at the end of both sub-parts making them able to stick to each other thus restoring the AC activity. Two plasmids were co-transformed in BTH101: pUT18-LZ containing the AC sub-part T18 fused with a LZ and pKT25-LZ_NLuc containing both the AC sub-part T25 fused with a LZ and the BioBrick PLac_NanoLuc.

800px-T--Grenoble-Alpes--Plasmid_pUT18LZ_pKT25LZ_NLuc.png

If there is a notable difference of luminescence between the free sub-parts and the target detection then it will mean that our reporter gene could work in our system. It will also show that the on/off switch of the transcription depending on cAMP is working.

The assay

Bacterial culture were induced with 0.5mM of IPTG at Optic Density 0.6. The subtract for Nano Luciferase (furimazine) was added as follow : for 50uL of bacterial culture in a well, 49uL of NanoGlo Assay Buffer and 1uL of NanoGlo Assay Substrat were added. All the measures are expressed in Relative Luminescence Units (RLU) in a NUNC 96 wells plate. Two different bacterial cultures (sample) were assessed each time in duplicate (except for the 24 hours condition). Blank was done with non-transformed BTH101 (RLU = 300) and subtracted from the measurements.

Results

T--Grenoble-Alpes--NLuc_Fig_1.png


The second well for sample 2 was removed because the assay substrate wasn’t added.


T--Grenoble-Alpes--NLuc_Fig_2.png


T--Grenoble-Alpes--NLuc_Fig_3.png

Conclusion

800px-T--Grenoble-Alpes--NLuc_Graph_Conclu.png

Measurement of the Nano Luciferase assays of the 3 conditions.

Those measurements highlight two major things with our reporter Biobrick: First, there is a significant luminescence gap between the assay without endogenous adenylate cyclase (grey) and with the free sub-parts (orange) which means that there is a significant leak of our reporter protein due to random occurrences between both T18 and T25. However here is a significant difference between the assay with or without LZ which means that when the target is present and brings the two parts together the gene (here Nano Luciferase) is overexpressed. With this BioBrick it is then possible to see the difference between both condition: if the two sub-parts are close and if they are free and so tell if the theoretical target is present or not. In realist conditions the difference will probably not be as flagrant as here because the LZ system is more efficient to bring the parts together than the assay done with the target and aptamer (see the full system).


User Reviews

UNIQb06c5fa916ba866b-partinfo-00000000-QINU UNIQb06c5fa916ba866b-partinfo-00000001-QINU