Revision as of 13:15, 15 October 2014

Project

Introduction

The 2014 EPFL iGEM team has been working on showing that biologically engineered organisms can detect and process signals quickly and efficiently. With this in mind, our team brought forward a novel idea: combining Protein Complementation techniques with biosensors to achieve fast spatiotemporal analysis of bacterial or yeast response to mechanical stimuli.

Our team explored this hypothesis by engineering two stress related pathways in E.Coli and S.Cerevisiae with in mind the development of a BioPad: a biological touchscreen consisting of a microfluidic chip, touch responsive bacteria, and a signal detector. Learn more about how the BioPad works !

The pathway engineered in E.Coli, the Cpx Pathway, is a two-component regulatory system responsive to envelope stress. A full description of the pathway is available here. In S.Cerevisiae we modified the HOG Pathway - a MAPKK pathway responsive to osmotic stress. For more information concerning the HOG Pathway click here.

Our project also includes an extensive microfluidics section. Our self designed chips helped us improve precision, safety, and quantification methods used throughout the project. To learn more about the microfluidic components of our project check out this link !

Last but not least, we designed a novel signal detector ! To make signal detection more practical we developed an automatised cheap tracking system made of a mini-computer (Raspberry Pi) and a mini-HD camera. More details concerning this the BioPad detector can be found here.

The Cpx Pathway

The natural function of the Cpx 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 Cpx two component regulatory system belongs to the class I histidine kinases and includes three main proteins:

How the BioPad works in E Coli

Our self-designed PDMS microfluidic chip, the BioPad, is made of hundreds of compartments representing "pixels." Each 30µm x 30µm x 3µm compartment contains a few layers of E. coli. When the surface of the chip is touched, a deformation of the chip - and thus of the compartments - leads to cellular membrane shear stress and protein aggregation/misfolding in the periplasm.
The aggregated/misfolded proteins are then sensed by the histidine kinase CpxA sensor, which 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 fluorescent protein fragments (split IFP1.4) via a 10-amino acid, 2x GGGGS flexible linker. This allows us to detect CpxR dimerization, synonymous periplasmic stress and touch. Moreover, the split protein fragments are reversible. Therefore, when stress is removed, CpxA changes conformation and dephosphorylates CpxR allowing it to dissociate. The signal is shutdown and darkness returns The BioTouch Detector (composed of an inexpensive CMOS called Raspberry Pi, a highly sensitive digital camera with appropriate light filters, and a light emitting source) 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 HOG Pathway

The HOG (High Osmolarity Glycerol) pathway is a MAPK (Mitogen activated protein kinase) pathway which yeast cells use to coordinate intracellular activities to optimise survival and proliferation in not only hyper-osmotic stress but also heat shock, nitrogen stress and oxidative stress. It is represented below.

The pathway includes five main proteins:

Sho1/Sln1 – Membrane proteins which are classed as STREs (STress Response Elements) which sense the stress and initiate the pathway
Ste11 – The MAPKKK which phosphorylates PBS2
PBS2 – The MAPKK which phosphorylates HOG1
HOG1 – The MAPK which localizes to the nucleus upon phosphorylation and induces target gene transcription

How we engineered the HOG pathway to make our BioPad

Our engineered yeasts cells can be loaded into a microfluidic chip made of small compartments able to contain a few layers of cells. When the surface of the chip is touched, it leads to a deformation of the chip - and thus of its compartments. Since the HOG pathway is reactive to turgor pressure, the pressure applied activates it. Upon induction of the pathway, which is a classical MAP kinase pathway, PBS2 – a MAPKK – is phosphorylated and binds HOG1 – a MAPK – and in turn phosphorylates it.

Therefore, we have fused these two kinases to split fluorescent and luminescent proteins, via a 13-amino acid flexible linker, by homologous recombination. This allows us to detect the phosphorylation of Hog1 by Pbs2 in response to osmotic pressure or touch. We have used split sfGFP and split Renilla luciferase tags on the C-terminals of both proteins.

As in the E.Coli, the split sfGFP is irreversible and was made to show the interaction between our two Pbs2 and Hog1 while we use the reversible split luciferase tags to assess the activation and inactivation of the pathway. In fact, when stress is removed, the signal should decline. The BioTouch Detector is programmed to identify and process the light position and can transmit the information to a computer.

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.

Team:EPF Lausanne/Overview

From 2014.igem.org