Team:EPF Lausanne
From 2014.igem.org
Our project in a nutshell
Summary of our project
The 2014 EPFL iGEM team has been working on showing that biologically engineered organisms 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 develop a BioPad: a biological TouchPad made of touch responsive bacteria in a microfluidic chip allowing the control of electronic devices. This was done by engineering the Cpx Pathway – a two component regulatory system responsive to periplasmic stress.
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;
Applications
The biopad is not the only application of our modified organisms and microfluidic devices.
Antibiotic screening device
Bacterial envelopes are often remodeled when encountering hosts. These changes lead to the synthesis of complex envelope structures that are important virulence factors. Improper assembly of these structures can harm the bacterial envelope and lead to Extracytosolic Stress. Bacteria counter the potential envelope stresses by downregulating these virulence factors.
Taking into consideration the close involvement of virulence factors and bacterial survival, the CpxA-R pathway has been shown to be a promising candidate as an antibiotic. When activated, the CpxA-R pathway activates a bacterial survival response which among other things, regulates and monitors the biogenesis of complex surface virulence factors such as pili/fimbiae and type III and type IV secretion systems. Equivalently, it has also been suggested that the CpxA-R system is involved in antibiotic mediated bacterial cell death. Our device would therefore allow us to detect and mesure activation of the CpxA-R system in real-time and thus assess the strength and influence of antiobiotics and antiobiotic candidates on the CpxA-R system.
The ultimate goal of this application would thus be to allow high-throughput screenings for antibiotic candidates enabling the removal of virulence factors from pathogenic bacteria. This would improve antibiotic treatment and serve as an “antibiotic complement”.
Another application to our BioPad organisms would be related to cancer. In modern research, tumor progression is fairly difficult to evaluate: most scientists rely on the size of a tumor to understand how developed it is. Our idea would be to integrate our engineered organisms within the tumor's cellular matrix (Matrigel) to allow researchers to be able to assess the progression of tumors by how luminescent the tumor is when a light emitting molecule -luciferin- is injected. This would allow scientists to reduce unnecessary animal sacrifices in tumor research.
RESULTS
Experiment 1: Promoter characterisation and folding ability of fused GFP to CpxR via 10 amino acid 2 x (GGGGS) flexible linker
Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus
Experiment 2: CheY/CheZ fused to split firefly/renilla luciferase, and full firefly/renilla luciferase characterisation
Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus
Experiment 3: CpxR dimerization & Dimerization Orientation
CpxR is the relay protein in the stress resonsive CpxAR two component regulatory system. It has been shown by split beta galactosidase assay that CpxR dimerizes when phosphorylated (activated) in yersinia pseudotuberculosis. Moreover, following other in vitro FRET studies, it was shown that E.Coli CpxR interacted with itself. We therefore hypothesised that dimerization would also be true in vivo in E.Coli.
To determine this, we built four constructs with the various possible orientations that the split IFP1.4 fragments could have with CpxR. As shown in the graph bellow, we successfully proved that CpxR dimerized in vivo and that dimerization led to close interaction of its C-terminus.
This finding is important as CpxR is part of the highly conserved OmpR/PhoB subfamily - especially for their C-terminus. This system could be used to study various other components of the OmpR/PhoB subfamily and thus lead to a new generation of highly senstitive and reactive biosensors.
As seen in the graph, induction of the signal was done at minute 24 (marked via a vertically spoted line). It is to be noted that the signal is immediate (3 fold increase in 2 minutes) and that the signal overall increased 30-fold.
The protocol for this experiment can be downloaded here.
Experiment 4: Signal induction by various concentrations of KCl & signal shutdown by centrifugation
Having found that KCl was a good signal inducer for our signal, we decided to characterise our biobrick by testing if the signal could be modulated by various concentrations of KCl and if we were able to remove the signal by centrifugation and medium change. To do so, we read our signal for 20 minutes without stress and then added KCl. At minute 144 we then centrifuged our cells and replaced the medium with PBS.
As seen in the figure above, we successfully showed that increasing concentrations of KCl led to stronger signals up to a saturation concentration of about 80 mM KCl. Moreover we were able to shut the signal down, thus proving the reversibility of our system.
Experiment 5: Microfluidic stuff ?
Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus
Experiment 5: Yeast stuff ?
Pellentesque habitant morbi tristique senectus et netus et malesuada fames ac turpis egestas. Vestibulum tortor quam, feugiat vitae, ultricies eget, tempor sit amet, ante. Donec eu libero sit amet quam egestas semper. Aenean ultricies mi vitae est. Mauris placerat eleifend leo. Quisque sit amet est et sapien ullamcorper pharetra. Vestibulum erat wisi, condimentum sed, commodo vitae, ornare sit amet, wisi. Aenean fermentum, elit eget tincidunt condimentum, eros ipsum rutrum orci, sagittis tempus lacus enim ac dui. Donec non enim in turpis pulvinar facilisis. Ut felis. Praesent dapibus, neque id cursus faucibus, tortor neque egestas augue, eu vulputate magna eros eu erat. Aliquam erat volutpat. Nam dui mi, tincidunt quis, accumsan porttitor, facilisis luctus, metus
MEET OUR TEAM
We are a group of 14 students from the faculties of Life, Biomechanical, and Computer Sciences, and are supervised by 2 EPFL professors, 1 Lecturer and 5 PhD students.