Difference between revisions of "Team:Utrecht/Design"

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<h3> BRET </h3>
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<h3> Bioluminescence Resonance Energy Transfer Pairs </h3>
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<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.
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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.</p>
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<p>To measure the concentration of ligand present, the activity of wild type and modified Tar receptors have to be measured. Therefore, we designed a Bioluminescence Resonance Energy Transfer (BRET) based sensor, inspired by a previously made chemotaxis BRET-pair (Cui et al. 2014). BRET is a technique where photons produced by a luciferase are used to excite a fluorophore. We opted to use Renilla luciferase (RLuc) since it produces photons that can excite eYFP. In addition, the substrate for this protein, coelenterazine, is permeable to the cell which makes it perfect for E. coli based bioassays. For energy transfer to occur, the RLuc and eYFP proteins must be less than 10 nm apart. Thus by measuring the ratio of light emitted by the two molecules, we can detect whether they are physically close, as can be seen in figure 2. </p>
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<p>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.</p>
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<img src="https://static.igem.org/mediawiki/2018/c/ce/T--Utrecht--2018--BRET_Assay--Main003.png" alt="BRET_Assay.png" width  = 60% height = auto>
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<figcaption>Figure 2: BRET-pair activity. A) When CheZ and CheY do not interact, the distance between Luciferase and eYFP is too large for Luciferase to excite eYFP. As a consequence, only Luciferase emits light. B) Upon interaction of CheZ and CheY, Luciferase and eYFP are in close proximity. eYFP is excited by photons produced by luciferase, leading to luminescence of eYFP.</figcaption>
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<p>By fusing the RLuc to CheZ and eYFP to CheY, the bioluminescence can be used as a proxy to demonstrate whether the chemotaxis proteins interact (Figure 3). When no ligand is bound to the Tar receptor, CheA is active and phosphorylates CheY. Because of this, the phosphorylated eYFP::CheY can interact with CheZ::Rluc, bringing RLuc and eYFP in close proximity of each other. This can be measured as a BRET signal when the RLuc substrate is added. Upon binding of a ligand to the Tar receptor, CheA becomes inactivated, ultimately leading to a decrease of the BRET signal. Importantly, the signal can be readily measured by using an affordable bioluminescence assay.</p>
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<center><img src="https://static.igem.org/mediawiki/2018/c/ce/T--Utrecht--2018--BRET_Assay--Main003.png" alt="BRET_Assay.png" width  = 60% height = auto></center>
 
  
 
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Revision as of 21:56, 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.


Chemotaxis: the optimal system to meet all requirements

We chose to base our biosensor on the bacterial chemotaxis system because it offers many opportunities to address the above considerations. Because it is a native bacterial system, it can be produced at low cost (requirement V) and responds rapidly to environmental input (requirement IV).

In their natural environment, the chemotaxis system is used by Escherichia coli bacteria to direct them towards the highest concentration of a ligand. This system consists of a receptor that can be modified to detect a wealth of compounds (requirement III), (Shuangyu Bi et al. 2016). Furthermore, this receptor can be methylated, causing the sensitivity of the receptor to decrease. Customizing the methylation states facilitates the ability to measure a set concentration range (requirement II).

When no ligand is bound to the chemotaxis receptor, it activates a signal transduction pathway of several proteins, including CheA, CheY, and CheZ (figure 1). First, CheA phosphorylates CheY, which subsequently translocates to the cell membrane where it binds the flagellar motor protein, thereby altering its rotational direction to a “running” state. After CheY is bound to the flagellar motor protein, it is subsequently dephosphorylated by CheZ. Upon ligand binding, the receptor is inactivated, leading to an inactivated pathway and a rotational direction of the flagellar motor protein in the “tumbling” state. Since the binding of CheY and CheZ is linked to ligand binding of the receptor, measurement of the binding of these proteins by using the BRET assay (described below) provides a quick and accurate indication of the concentration of ligand present (requirement I and II).

Chemotaxis pathway modifications to address all considerations

To create a biosensor meeting all five listed requirements, the E. coli chemotaxis pathway was customized using a three step approach. First, we set up an assay to verify the extent to which one of the most important chemotaxis receptors, the Tar receptor, could be customized. This enables accurate detection and the possibility to detect diverse ligands (requirement III). Next, a method establishing the ability to measure the activity of this pathway was implemented, using a Bioluminescence Resonance Energy Transfer (BRET)-pair. BRET provides a clear and easily measurable detection signal (requirement II and IV). The last step of the approach facilitates accurate detection at different concentrations, by mimicking receptor methylation. This facilitates detection of different ligand concentrations (requirement I).

Bioluminescence Resonance Energy Transfer Pairs

To measure the concentration of ligand present, the activity of wild type and modified Tar receptors have to be measured. Therefore, we designed a Bioluminescence Resonance Energy Transfer (BRET) based sensor, inspired by a previously made chemotaxis BRET-pair (Cui et al. 2014). BRET is a technique where photons produced by a luciferase are used to excite a fluorophore. We opted to use Renilla luciferase (RLuc) since it produces photons that can excite eYFP. In addition, the substrate for this protein, coelenterazine, is permeable to the cell which makes it perfect for E. coli based bioassays. For energy transfer to occur, the RLuc and eYFP proteins must be less than 10 nm apart. Thus by measuring the ratio of light emitted by the two molecules, we can detect whether they are physically close, as can be seen in figure 2.

BRET_Assay.png
Figure 2: BRET-pair activity. A) When CheZ and CheY do not interact, the distance between Luciferase and eYFP is too large for Luciferase to excite eYFP. As a consequence, only Luciferase emits light. B) Upon interaction of CheZ and CheY, Luciferase and eYFP are in close proximity. eYFP is excited by photons produced by luciferase, leading to luminescence of eYFP.

By fusing the RLuc to CheZ and eYFP to CheY, the bioluminescence can be used as a proxy to demonstrate whether the chemotaxis proteins interact (Figure 3). When no ligand is bound to the Tar receptor, CheA is active and phosphorylates CheY. Because of this, the phosphorylated eYFP::CheY can interact with CheZ::Rluc, bringing RLuc and eYFP in close proximity of each other. This can be measured as a BRET signal when the RLuc substrate is added. Upon binding of a ligand to the Tar receptor, CheA becomes inactivated, ultimately leading to a decrease of the BRET signal. Importantly, the signal can be readily measured by using an affordable bioluminescence assay.