Difference between revisions of "Team:Utrecht/Design"

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<h3> BRET </h3>
 
<h3> BRET </h3>
 
<p>Bioluminescence Resonance Energy Transfer (BRET) is a technique in which a fluorophore is being excited by the light produced by a luciferase. This method only works when the luciferase emits light with a wavelength that lies within the excitation spectrum of the fluorophore. In addition, the fluorophore and the luciferase need to be less than 10 nm apart from each other. By fusing the luciferase, and the fluorophore to two different proteins A, and B, one can demonstrate whether these proteins interact by measuring the light intensity produced by both proteins.
 
<p>Bioluminescence Resonance Energy Transfer (BRET) is a technique in which a fluorophore is being excited by the light produced by a luciferase. This method only works when the luciferase emits light with a wavelength that lies within the excitation spectrum of the fluorophore. In addition, the fluorophore and the luciferase need to be less than 10 nm apart from each other. By fusing the luciferase, and the fluorophore to two different proteins A, and B, one can demonstrate whether these proteins interact by measuring the light intensity produced by both proteins.

Revision as of 21:47, 12 October 2018

In order to achieve our goal of creating a biosensor for the detection of pharmaceutical pollution, a structured approach to our design process was necessary. Determining the requirements for such a biosensor to be functional, and finding the right opportunities and systems to implement these requirements in, resulted in our choice for a three-step approach. On this page, you will find explanations of these three different components that constitute our project, as well as the reasoning behind our choice for a chemotaxis-based biosensor.

Biosensor requirements according to experts and stakeholders

Our stakeholder feedback sessions pointed us in the direction of developing a biosensor for detecting pharmaceuticals. Along this journey, we incorporated the following requirements of our biosensor based on stakeholder feedback:

  1. accurate detection at different concentrations;
  2. clear and easily measurable detection signal;
  3. possibility for detecting diverse ligands;
  4. rapid signaling;
  5. low-cost system.

BRET

Bioluminescence Resonance Energy Transfer (BRET) is a technique in which a fluorophore is being excited by the light produced by a luciferase. This method only works when the luciferase emits light with a wavelength that lies within the excitation spectrum of the fluorophore. In addition, the fluorophore and the luciferase need to be less than 10 nm apart from each other. By fusing the luciferase, and the fluorophore to two different proteins A, and B, one can demonstrate whether these proteins interact by measuring the light intensity produced by both proteins. In order to measure the activity of both wild type, as well as customized Tar receptors we designed a Bioluminescence Resonance Energy Transfer (BRET) sensor. This sensor consists of a Renilla luciferase (RLuc), and eYFP fused to the chemotaxis proteins CheZ, and CheY respectively. eYFP::CheY can be excited by the light produced by CheZ::RLuc, which has a wavelength of 480 nm, since the excitation spectrum of eYFP ranges from 400 to 550 nm.

When no ligand is bound to the receptor, eYFP::CheY is phosphorylated by CheA and can bind to CheZ::Rluc. As a result of this interaction, the distance between RLuc and eYFP is small enough to get a BRET signal when the RLuc substrate, coelenterazine, is added. Upon binding of a ligand to the Tar receptor, CheA becomes inactivated. As a result, the levels of phosphorylated eYFP::CheY drop and a decrease in the strength of the BRET signal is observed.

BRET_Assay.png