Applications

Basic Sciences Related

Protein complementation techniques & biosensors

Biosensors have been extensively characterized in previous iGEM projects. They consist of cells engineered to respond to a given stimulus through an intracellular signalization cascade, which often results in the transcription of a reporter gene. In most cases, the corresponding reporter protein is fluorescent or luminescent and its synthesis leads to a measurable output (ref).

Protein complementation techniques allow one to fuse complementary fragments of a split reporter protein to two other proteins of interest that interact together at a certain point. When the interaction happens, the fused reporter fragments are physically brought together, which reconstitutes the activity of the reporter protein (ref). By choosing two proteins which interaction is caused by a certain stimulus, one engineers a biosensor that responds much faster than traditional transcriptional systems – in the range of minutes rather than hours! Indeed, the reporter protein does not need to be synthesized but is already present in the cell and can activate as soon as its full structure is reconstituted.

In research, protein complementation studies are mostly used to validate protein interaction in the context of signal cascades and other pathways (ref). We think that their use as reporter systems in biosensors can have a broad range of applications in which the reporter signal needs to be measured quickly. One can think for example of water safety biosensors, in which one wants to know if water is contaminated or not before drinking it.

The great advantage of such a system is its modularity as it can be adapted to potentially any stimulus-responsive pathway in which two proteins are to interact at a certain point. It is also possible to choose between several split reporter proteins. The ones existing so far are fluorescent proteins – such as GFP, YFP or IFP – and luminescent proteins – such as Renilla or Firefly luciferases. But one could also imagine engineering split chromoproteins (for example, the ones developed by the 2009 Cambridge team) to have a device that changes colour straight away in response to a certain stimuli.

Microfluidics as an interface for the study of biosensors

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Enlightening the HOG pathway, but not only

The yeast part of our project is one of the first to study the HOG pathway directly with split complementation. Our PBS2 and HOG1 tags are thought to allow extensive characterization of the pathway in a very direct manner. The MAPKK-MAPK interaction could be quantified in terms of reaction time and sensibility to different stresses and the results would probably apply to many other pathways. Using our system, the pathway activation can indeed be triggered and verified very easily using the split luciferase strains, while localization of the interacting proteins can be observed with the GFP tag strains and single cell imaging.

Furthermore, the PBS2-HOG1 pathway is analogous to another pathway in mammalian cells, which involves the MEK5 and ERK5 proteins. Fluorescent mice are a thing of the past, we would now have touch-sensitive luminescent mice! While that might be a bit extreme as a potential application, expanding the project to mammalian cells using homologous recombination in mouse cells is not as far-fetched as it sounds. It would give an indication on how mammalian cells sense different stresses, since we could test them separately in an in vitro environment. This would be very useful since it is not yet clear how each pathway is regulated depending on different conditions and our system would provide a basis for mass screening.

Study of the transcription time

Our project also introduces a new way of studying the relationship between gene transcription and the corresponding activating signal. The idea would be to combine the fusion of split complementary fragments to dimerizing transcription factors, and the introduction of reporter constructs under promoters sensitive to the same transcription factor. One could study the relationship between these two signals and notably measure the time elapsed between binding of the transcription factor to the promoter and effective transcription of the reporter gene. Especially in yeast, induction of genes is much less understood than in bacterial cells and much more difficult. Our system would be very useful in controlling the intracellular environment of yeast cells, which would help the advance of synthetic biology in non-bacterial cells.

Applied Sciences Related

Cheap, fast, efficient, and accurate antibiotic screening system

The 2014 EPF Lausanne iGEM team engineered the CpxA-R pathway to develop its BioPad. The CpxA-R pathway responds to periplasmic stress via the presence of misfolded/aggregated proteins in the periplasm. Our team hypothesised that the presence of antibiotics would lead to a certain degree of protein misfolding/aggregation and thus would activate the signal. Since our device emits fluorescence upon periplasmic stress, our system could be used to quantify the strength of an antibiotic in a CpxA-R dependent manner. Combined to a microfluidic chip, this system could provide the scientific community with a cheap, fast, efficient, and accurate antibiotic screening system. This could result in easily quantifiable high-throughput screenings for antibiotic candidates.

Antibiotic Complement

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. The CpxA-R pathway, used by the 2014 EPF Lausanne, turns out to be envolved in this process. In fact when turned on, the CpxA-R pathway activates a bacterial survival response which among other things, down regulates the biogenesis of complex surface virulence factors such as pili/fimbiae and type III and type IV secretion systems.
Given this information, our device could lead to the discovery of an "antibiotic complement" enabling the removal of virulence factors from pathogenic bacteria to improve the efficiency of current antibiotics. Our vision of this novel form of antibiotic would be as a supplement to be taken with current antibiotics to improve the efficiency of treatment.

Tumor progression evaluation

A possible application of the organisms developped through this project would be related to cancer. In modern research, in vivo tumor progression in experimental animals is fairly difficult to evaluate: most scientists rely on the size of a tumor to get an idea of 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 much fluorescence the tumor emits. This would allow scientists to reduce unnecessary animal sacrifices in tumor research.

Tumor sensing

Further expanding on the mammalian application for the yeast part of our project, we could potentially create stress-sensing mice. As mentioned above, detection of a tumor is not the simplest of tasks. We would apply our system to whole mice, meaning that all their cells would be stress-sensors. At the injection of coelenterazine, the substrate for the Renilla Luciferase, the most stressed cells would luminesce meaning that we would easily detect the most stressed regions of the animal in question without hurting the animal or affecting its metabolism.

References

1: Michnick, S., Tchekanda, E., & Sivanesan, D. (2014, April 20). An infrared reporter to detect spatiotemporal dynamics of protein-protein interactions. Nature Methods, 6-6.