Team:EPF Lausanne/Overview

From 2014.igem.org

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<h1 class="cntr">Project</h1>
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                    <h2 class="section-heading">How the BioPad works</h2>
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The process by which a signal is detected upon touch starts of from our self-designed PDMS microfluidic chip: the BioPad. The BioPad is made of hundreds of compartments that represent the "pixels" of our pad. Each chamber has dimensions of 30µm x 30µm x 3µm allowing the BioPad to have single layers of E.Coli. When the surface of the chip is touched, a deformation of the chip - and thus of the chambers - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm. The aggregated/misfolded proteins are then sensed by the sensor histidine kinase CpxA that auto-phosphorylates and transfers its phosphate to its corresponding relay protein CpxR. Upon phosphorylation, CpxR homo-dimerizes. Our engineered bacteria contain CpxR proteins fused to split reversible fluorescent or luminescent protein fragments (IFP1.4 or firefly luciferase) via a 10 amino acid 2 x GGGS flexible linker. Therefore our engineered bacteria allow us to detect CpxR dimerization, synonymous periplasmic stress and touch. Then, a self built detector made of a raspberry pi, an inexpensive CMOS, and a couple of lenses, identifies and processes the position of the light/fluorescence emitted by the BioPad. This information about the position of the light relative to chip is then used to control the associated electronic device.
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Our biological Touch Pad will allow to control electronic devices by emitting light at the specific location where the Pad has been touched. Light emission is possible by engineering reporter proteins such as the firefly Luciferase, the Renilla luciferase, the Infrared fluorescent proteins, and even the superfolder GFP.
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We split the reporter proteins and fused them to an E.Coli endogenous protein involved in the regulation of extracytoplasmic stress. The protein of interest is CpxR, a component of the CpxA-CpxR two-component regulatory system.<br /><br /> -->
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On top of developing the biological components allowing fast response to stimuli, we engineered a small, cheap and easy to use “microscope” mainly made of a small camera to detect the position of the emitted light, process the information and instruct the associated electronic device that the user is touching the BioPad a given position.
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  <h2 class="section-heading">The CpxA-R Pathway</h2>
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The natural function of the CpxA­-CpxR two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses.
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The CpxA-­CpxR two component regulatory system belongs to the class I histidine kinases and includes three main proteins:
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  <dt>CpxA</dt>
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  <dd>an integral inner­-membrane sensor kinase, which activates and auto­phosphorylates when sensing misfolded proteins in the E.Coli periplasm. CpxA transduces its signal through the membrane to activate the cytoplasmic CpxR response regulator by a phosphotransfer reaction.</dd>
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  <dt>CpxR</dt>
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  <dd>CpxA’s corresponding cytoplasmic response regulator belongs to the OmpR/PhoB family of winged­helix­turn­helix transcriptional response regulators and is phosphorylated by CpxA in the presence of extracytoplasmic stresses. Phosphorylation induces CpxR’s homo­dimerization, and activation as a transcription factor. Phosphorylated CpxR then binds to the promoters of genes coding for several protein folding and degradation factors that operate in the periplasm.</dd>
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  <dt>CpxP</dt>
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  <dd>an inhibitor of CpxA that we suspect to actively compete with misfolded proteins (CpxP is a chaperone).</dd>
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  <h2 class="section-heading">Engineering: CpxR and split complementation techniques</h2>
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The main component that we wish to engineer is CpxR. It has been reported that the protein homo-dimerizes upon activation. We thus plan to use fused split proteins of fluorescent and bioluminescent nature to detect its activation. <br /><br />
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table
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As a preliminary step, we used two fluorescent proteins sfGFP and IFP to characterisation CpxR. Split sfGFP (superfolder GFP) is an irreversible split system which will be used to prove the dimerization of CpxR. Split IFP on the other hand (Infrared Fluorescent Protein) is a reversible split system which will be used to understand the spatio-temporal dimerization of CpxR and thus allow us to better understand the On/Off mechanism of this system.<br /><br />
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To achieve our final goal, we will engineer split bioluminescent proteins: Firefly and Renilla split Luciferases. These constructs will be the main component of our system. When fused to CpxR, we are expecting to witness emission upon touch.
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  <h2 class="section-heading">Engineering: Microfluidic chip interface</h2>
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Our specially designed microfluidic chip, hereafter known as BioPad Chip, will allow easy and accurate induction of fluorescent or bioluminescent signals. The chip is made up of thousands of compartments – representing the pixels of our device ­- of the height of a bacteria. The BioTouch Chip thus allows the effective trapping and induction of stress onto our engineered bacteria. <br /><br />
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<h2 class="section-heading">Engineering: The BioPad Detector</h2>
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The signals induced by the BioTouch Chip are then processed by our self designed detection system: the BioTouch Detector. The BioTouch Detector is mainly made of a cheap computer (Raspberry Pi), a highly sensitive digital camera with appropriate light filters, and a light emitting source. The BioTouch Detector locates signals from various sources (infrared fluorescence, green fluorescence and luminescence), processes them and sends back the relative positions of the signals with respect to the BioTouch Pad. Thanks to this position, we are able to extract information such as giving a computer operating system that the position represents the position of the mouse on a screen, that the well at the given position is a suitable antibiotic candidate, or that a gene of interest has been activated. We therefore effectively control a computer or any other electronic device through a living interface: the BioTouch Pad.
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Revision as of 11:46, 28 September 2014

Project

How the BioPad works

The process by which a signal is detected upon touch starts of from our self-designed PDMS microfluidic chip: the BioPad. The BioPad is made of hundreds of compartments that represent the "pixels" of our pad. Each chamber has dimensions of 30µm x 30µm x 3µm allowing the BioPad to have single layers of E.Coli. When the surface of the chip is touched, a deformation of the chip - and thus of the chambers - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm. The aggregated/misfolded proteins are then sensed by the sensor histidine kinase CpxA that auto-phosphorylates and transfers its phosphate to its corresponding relay protein CpxR. Upon phosphorylation, CpxR homo-dimerizes. Our engineered bacteria contain CpxR proteins fused to split reversible fluorescent or luminescent protein fragments (IFP1.4 or firefly luciferase) via a 10 amino acid 2 x GGGS flexible linker. Therefore our engineered bacteria allow us to detect CpxR dimerization, synonymous periplasmic stress and touch. Then, a self built detector made of a raspberry pi, an inexpensive CMOS, and a couple of lenses, identifies and processes the position of the light/fluorescence emitted by the BioPad. This information about the position of the light relative to chip is then used to control the associated electronic device.

The CpxA-R Pathway

The natural function of the CpxA­-CpxR two component regulatory system in bacteria is to control the expression of ‘survival’ genes whose products act in the periplasm to maintain membrane integrity. This ensures continued bacterial growth even in environments with harmful extracytoplasmic stresses. The CpxA-­CpxR two component regulatory system belongs to the class I histidine kinases and includes three main proteins:

CpxA
an integral inner­-membrane sensor kinase, which activates and auto­phosphorylates when sensing misfolded proteins in the E.Coli periplasm. CpxA transduces its signal through the membrane to activate the cytoplasmic CpxR response regulator by a phosphotransfer reaction.
CpxR
CpxA’s corresponding cytoplasmic response regulator belongs to the OmpR/PhoB family of winged­helix­turn­helix transcriptional response regulators and is phosphorylated by CpxA in the presence of extracytoplasmic stresses. Phosphorylation induces CpxR’s homo­dimerization, and activation as a transcription factor. Phosphorylated CpxR then binds to the promoters of genes coding for several protein folding and degradation factors that operate in the periplasm.
CpxP
an inhibitor of CpxA that we suspect to actively compete with misfolded proteins (CpxP is a chaperone).

Engineering: CpxR and split complementation techniques

The main component that we wish to engineer is CpxR. It has been reported that the protein homo-dimerizes upon activation. We thus plan to use fused split proteins of fluorescent and bioluminescent nature to detect its activation.

table As a preliminary step, we used two fluorescent proteins sfGFP and IFP to characterisation CpxR. Split sfGFP (superfolder GFP) is an irreversible split system which will be used to prove the dimerization of CpxR. Split IFP on the other hand (Infrared Fluorescent Protein) is a reversible split system which will be used to understand the spatio-temporal dimerization of CpxR and thus allow us to better understand the On/Off mechanism of this system.

To achieve our final goal, we will engineer split bioluminescent proteins: Firefly and Renilla split Luciferases. These constructs will be the main component of our system. When fused to CpxR, we are expecting to witness emission upon touch.

Engineering: Microfluidic chip interface

Our specially designed microfluidic chip, hereafter known as BioPad Chip, will allow easy and accurate induction of fluorescent or bioluminescent signals. The chip is made up of thousands of compartments – representing the pixels of our device ­- of the height of a bacteria. The BioTouch Chip thus allows the effective trapping and induction of stress onto our engineered bacteria.

Engineering: The BioPad Detector

The signals induced by the BioTouch Chip are then processed by our self designed detection system: the BioTouch Detector. The BioTouch Detector is mainly made of a cheap computer (Raspberry Pi), a highly sensitive digital camera with appropriate light filters, and a light emitting source. The BioTouch Detector locates signals from various sources (infrared fluorescence, green fluorescence and luminescence), processes them and sends back the relative positions of the signals with respect to the BioTouch Pad. Thanks to this position, we are able to extract information such as giving a computer operating system that the position represents the position of the mouse on a screen, that the well at the given position is a suitable antibiotic candidate, or that a gene of interest has been activated. We therefore effectively control a computer or any other electronic device through a living interface: the BioTouch Pad.

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