Part:BBa_K3128001:Experience

Team Grenoble Alpes 2019

Applications of BBa_K3128001

More information : Grenoble-Alpes 2019

Our detection system is based on the use of a BACTH. The point is to conditonally induce the expression of the gene upon interaction of the two sub-parts of Adenylate Cyclase (AC) are physically close, which only occurs when the target is present in the sample and detected by our NanoDrop system.
The re-constitution of AC then enables cAMP production, which will activate a CAP dependent promotor allowing the transcription of the downstream gene.
In order to perform the assay we needed to use an AC deficient bacteria strain (BTH101) that do not produce endogenic cAMP. This property prevents any transcription from CAP dependant promoter such as lactose promoter.
For the choice of the promoter, we decided to use the lactose promoter (a CAP dependent promoter) and we have demonstrated its repression in absence of cAMP (in the AC deficient bacterial strain), thus preventing any transcription of the following gene : the reporter one (see table and figure 1). To resume, the expressed/overexpressed of the gene is under the control of cAMP For a good sensitivity of our system, statistically different signals have to be recorded on negative sample (no AC reconstitution) and positive sample (constitutive reconstitution of sub-parts of the AC).

To prove that the reporter gene efficiently works in our system different conditions were tested.
First the leak of our reporter in absence of cAMP was measured by transforming the plasmid containing the BioBrick PLac_NanoLuc in BTH101.

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.


At condition imitating the target's recognition was tested. Leucine-Zipper (LZ) were used to mimic 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 in absence of target thus restoring the AC activity.
Two plasmids were co-transformed in BTH101: pUT18-LZ containing the AC sub-part T18 fused with a LZ at the C terminal and pKT25-LZ_NLuc containing both the AC sub-part T25 fused with a LZ at the C terminal and the BioBrick PLac_NanoLuc.

If there is a significant difference of luminescence between the free sub-parts condition and the target detection imitating condition then it will demonstrate that our reporter gene is working in our system.
It will also show the cAMP dependent on/off transcription switch.

The assay

Bacterial culture were induced with 0.5 mM of IPTG at an Optical Density of 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.
The bioluminescence expressed in Relative Luminescence Units (RLU) were recorded in a black NUNC 96 wells plate.
Two different bacterial cultures (sample) were assessed per experiment in duplicate (except for the 24 hours condition).
Blank was done with non-transformed BTH101 (RLU = 300) and subtracted to each measurements.

Results

The second well for sample 2 was removed because the substrate was omitted.
Figure 1 : recombinant bacteria containing the NanoLuciferase gene without endogenous adenylate cyclase (grey).
Bioluminescence was recorded at different time points post induction.



Figure 2 : recombinant bacteria containing the pUT18 and pKT25_NLuc plasmids (orange).
Bioluminescence was recorded at different time points post induction.



Figure 3 : recombinant bacteria containing the pUT18-LZ and pKT25-LZ_NLuc plasmids (blue).
Bioluminescence was recorded at different time points post induction.

Conclusion

Figure 4 : Recombinant bacteria containing the NanoLuciferase gene without (grey) or with the pUT18 and pKT25_Nluc plasmids (orange) or with the pUT18-LZ and T25-LZ_Nluc AC sub-parts were culture. Bioluminescence was recorded at different time points post induction..

Those measurements highlight two major points:
First, the luminescence produced by the strain without AC (grey) is low and stable over time. It can be considered as the background of the system.
In contrast the Bioluminescence recorded in the recombinant bacteria containing the free sub-parts of AC (orange) and LZ mediated reconstituted AC (Blue) showed a 1 to 10 order of magnitude increase and evolved over time to reach 5.9 E+ 07 +/- 4.9E+06 and 1.6 E+08 +/- 1.5 E+06 respectively 24 hours after induction. These data suggest that T18 and T25 randomly reconstitute in the cytoplasm leading to cAMP production and expression of the NanoLuc reporter gene.

However, LZ mediated interaction of the two sub-parts of AC leads to a significant* increase of NanoLuciferase overexpression when compared with the random reconstitution.
* A T test was done and led to a p-value < 0.05.

Thanks to this BioBrick it is then possible to measure the difference between both conditions: random or LZ mediated reconstitution of AC. As a consequence these system could be adapted to our NeuroDrop project for the detection of extracellular biomolecule through the biosensor system conjugated to aptamers described elsewhere (see the full system).

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