Our project in a nutshell

Summary of our project

The 2014 EPFL iGEM team has been working on showing that biologically engineered contexts can detect and process signals in fast and efficient ways. With this in mind, our team worked on bringing forward a novel idea: combining Protein Complementation techniques to Biosensors to achieve fast spatiotemporal analysis of bacterial response to stimuli.

As a proof of concept of this idea, we aimed to engineer the Cpx Pathway – an E.Coli endogenous two component regulatory system responsive to periplasmic stress – to develop a BioPad: a biological TouchPad made of touch responsive bacteria in a microfluidic chip allowing the control of electronic devices .

Why a BioPad ?

The biological concepts behind the BioPad project have applications both in basic and applied sciences. From a purely scientific perspective, the ideas introduced and implemented by our project are novel and promising for future applications. The BioPad is also an attractive concept that is tangible for the general public and will allow people to look at synthetic biology in a different way. Hence, the combination of novel biological concepts, a cool idea, and the community awareness that our project provides, makes the BioPad project perfect for iGEM !

The BioPad's applications in a nutshell

With respect to basic sciences, our system serves as a good proof that protein complementation techniques are suitable for applications in the context of biosensors – especially for two component regulatory systems. The introduction of the split IFP1.4 into the registry will allow future iGEM and research teams to take advantages of the reversibility and precision of this protein. Moreover, our work on the Cpx pathway will allow future iGEM teams to make us of other members of the OmpR/PhoB subfamily as well as other two-component regulatory systems in new ways.

As for applied sciences, the potential uses of the BioPad include the delivery of a cheap, fast, efficient, and accurate antibiotic screening systems enabling an easy way to quantify how antibiotics affect the periplasm in gram negative bacteria; the BioPad project could also be the source of an "antibiotic complement" drug allowing could also provide a new way to study genes by allowing the examine the relationship between genes and their corresponding activating signals;

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.

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.