Team:Leeds/Project

Our Project

What is MicroBeagle I hear you cry?

Well, MicroBeagle is a E.coli cell that has been modified so that it can help “hunt down” pathogens. Just like a Beagle, the hunting dog, searches for, finds, and indicates the presence of rabbits and hares, our MircoBeagle finds pathogens, detecting them through physical binding, and lights up to indicate their presence! By utilising the Cpx pathway, which responds to membrane stress (which we hypothesize would be induced by the binding event) and GFP (as a reporter) we hope to create a new biosensor that gives results that can be seen with the naked eye.

Our MicroBeagle project is laying groundwork towards the development of a modular, general platform for pathogen detection. What makes Leeds iGEM's MicroBeagle distinctive to previous strategies used for analyte detection within iGEM is that we use physical binding as our detection mode. This should be well suited to detecting a wide range of pathogen targets, as tailored target-binding proteins or peptides can be displayed from the cell surface, allowing specific binding to the pathogen of interest.

Why is the MircoBeagle a useful device?

There could be many different possible uses for our device, as the pathogen detection mechanism we are using is modular and can easily be tailored to individual needs. This is achieved by swapping out the gene coding the cell surface-displayed binding domain to suit your need. We envision the MircoBeagle being used for testing blood samples, detection of heavy metals and, in the application that is our main inspiration, detection of pathogens in water samples.

Why Water Testing is so important

Detecting pathogens in water is a really important application as 3.4 million people a year die from water related diseases. 768 million people in the world do not have access to safe water. This is roughly one in ten of the world's population, the majority of which is caused by faecal contamination: poor sewage systems that flow back into the water supply. The issue is actually more complex and subtle than just direct health problems, the knock on economic problems that result from poor sanitation are phenomenal. Lack of water, sanitation and hygiene costs Sub-Saharan African countries more in lost GDP than the entire continent gets in development aid. The figures are shocking and we as a team want to create something that may, in the future, help to reduce those numbers. While many organization are creating new, appropriate water purification devices and systems, we realize these will only be as good as the measurement technologies available to test their performance will allow. This bottleneck is particularly critical in infrastructure-poor areas with no access to the molecular biology labs typically used to assay water-borne pathogens. While many teams around the world are seeking new ways of transporting chassis and bio-devices in facile, safe, and reliable manners (e.g., through capsules or spores), we felt that efforts to develop modular pathogen detection systems were lacking. Once effective transport strategies are established, we hope to have a detection system ready-to-go to employ in on-site water testing and diagnostics. To address this issue using a novel synthetic biological approach, the MircoBeagle was born!

Below is a summary of our Device development strategy...

Phase I

We split our project into self-contained phases, to help better organise ourselves, but also lay out a road map for our own future work within iGEM and beyond. Our two devices include a device for activating a reporter in response to membrane stress (Device 1) and a device for binding to a physical target at the cell surface and thereby activate the membrane-stress-reporter (Device 2).

Device 1

Device 1 is a very simple modification of the Cpx promoter in E. coli to produce a fluorescent reporter when the cell is under membrane stress. We will then characterise and test the insertion following the work by [http://www.ncbi.nlm.nih.gov/pubmed/11830644 Otto & Silhaevy 2001] in which glass beads were used to induce hydrophobic membrane stress. What we would hope is that when we add our cells to a vial containing similar hydrophobic beads we can detect fluorescence emissions confirming that our device is working as we had hoped. We know that membrane stress can be caused by many stimuli, so we have also looked at the effect different membrane stresses cause on the florescence achieved; these membrane stresses include, pH, temperture and detergent concentration.
Bio bricks we used to assemble Device 1 are: BBa_K135000 pCpxR promoter responds to membrane stress BBa_K081012 Green fluorescent protein generator. Contain ribosome binding site and terminator

Si4 Binding Peptide

Simultaneously, we will be developing Device 2 that is suited for physical attachment and detection of particles. This will work using Ice Nucleation Protein (INP) to display a oligo-peptide of our choice on the outer-membrane of our E. coli, initially this will be a peptide capable of binding silica beads allowing us to create a model system of pathogen detection. INP is a transmembrane protein that expresses any sequence that is placed on the C-terminus of its gene on the outer surface of the cell. Check out our Lab results page to see the characteristaion results for the Si4 binding domain.

The Biobricks we used for this Device are: BBa_K081012 for the Green fluorescent protein generator which contain a ribosome binding site and terminator. BBa_K523008 is the Ice nucleation protein any sequence added to the C-terminus of the INP will be transported to cell membrane and be displayed on the surface of the cell. BBaa_B0015, which is a double terminator, BBa_B0034 a strong ribosome binding site and finally

Phase II

Phase II takes the products of Phase I to the next level, and begins to look at integration of the Si4 binding peptide and Device 1 to give a new device; Device 2.

Device 2

Device 2 brings together BS1 and BS2 in one plasmid; the genes that code for the INP and the Si4 binding domain are now located on the same plasmid as the CpxP promoter and the GFP generator. The effect of this is that when the silca bead binds to the Si4 binding domain connected to the INP protein and causes surface stress the CpxP promoter will be induced. This will cause GFP to be produced and this will act as a visible signal that the 'pathogen' has been detected.
This is the final MircoBeagle device and has the potential to be made into many different pathogen detectors. This can easily be done by swapping out the gene coding for the binding moiety next to the INP gene. Once Biosystem 2 has been suitably characterised and all bugs fixed, the next step will be to swap the silca binding domain for a binding domain that binds to a particular pathogen.

The Cpx Pathway

This project is highly dependent upon a naturally occuring pathway in E.Coli called the Cpx pathway. It is associated with the regulation of periplasmic membrane stress and the misfolding of surface proteins. We may need to fine tune MicroBeagle for different applications, so by understanding the way the pathway is regulated, we stand a better chance of controlling the exact response we want. This may be done in a number of ways, from utilisation of an off-switch regulator, CpxP, to additonal pathways used to create a bio-logic gate or even by optimisation of buffer solution.

A brief explanation of the Cpx Pathway

The Cpx pathway responds to various membrane stress; it is a two component signal transduction pathway. The first component is CpxA, an inner membrane protein which, when bound to CpxP (an inhibitor molecule), in inactive. When CpxP is unbound from CpxA, CpxA autophosphorylates itself. A kinase enzyme then phosphorylates the CpxR, the second component in the pathway, which then binds to the Cpx promoter and activates transcription. There is still a lot of debate regarding this pathway and many different theories but it is thought that membrane stress causes the misfolding of proteins (pili being the main contributor) causes misfolded subunits to accumulate in the periplasm and titrate the CpxP inhibitor molecule off the CpxA and activate the pathway.