Part:BBa_K3128004:Experience

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Applications of BBa_K3128004

Complete article : (link)

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. The produced cAMP will then activate CAP dependent promoter allowing the transcription of the following gene. 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. 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 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.

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.

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.

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.

For 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 Substrate were added then wait 5 minutes before the measurement. 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). Blank was done with BTH101 non-transformed (RLU = 300) and subtracted from the measurements.

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

Conclusion : 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. 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).

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