Future Applications of Research

We believe that the initial stages of the Biosensor have been complete and will lead to many new and exciting developments in the future. The general idea is that once the surface protein has been expressed, it will be able to interact with Dermo and produce a signal.

As for this year we our efforts working with a Bovine-Galectin have come the furthest and are ready to move into these next steps.  Additionally in future years we hope to incorporate CvGal1 into the constructs, an effort we were not able to accomplish due to lack of time and troubleshooting. 

Signaling Approaches:

1). Genetic engineering approach -Downstream Signaling Cascade

One of the strategies we hope to pursue is developing or incorporating a downstream signaling cascade into the cell strain. In this effort, once the E. coli came in contact with the Dermo it would then set off a transcription factor, ultimately producing a signal.  Multiple approaches have been researched, including a Cpx membrane stress cascade, a split GFP cascade, and others.

The Cpx two component system reacts naturally to membrane stress. When the much larger Dermo is bound to the surface receptor expressed on E. coli, the bacterial cell membrane would become deformed. In turn, this would transmit a signal to the nucleus via the Cpx system that could be coupled to GFP production. This assay would require florescence microscopy to detect Dermo in solution.

Another option is for a split GFP construct.  The idea is two different constructs containing the ligand are present in the cell, each with a different fraction of the GFP gene. When Dermo comes in close contact with the cell, multiple ligands on the cell would bind to the receptors on Dermo, causing the membrane to pinch or come together. This would trigger a spontaneous chance that two complementary pieces of GFP in close contact would bind, producing a fluorescent signal.  This would also need to be observed using microscopy.

One of the ultimate goals from using the downstream signaling approach is to develop a “Kamikaze Bacteria”. By this we hope that the bacteria would respond to the presence of Dermo by eliminating it, possibly through killing itself. Similar work has previously been done with bees and colony collapse.

2). Microfluidics Approach- Size Based Cell sorter

Another avenue we plan to pursue is a microfluidics based detection approach.  The overall concept would rely on the fact that E.coli cells are much smaller than Dermo cells. By creating a microfluidic device that sorts based on size, E.coli would travel into one output, whereas the Dermo and Dermo+E.coli complexes would travel into another output. Furthermore if the E.coli cells are fluorescently stained, analysis of the larger size fraction would indicate whether any E.coli cells are bound, confirming the presence of Dermo.  While further differentiations based on size between bound and unbound Dermo may not be possible due to growth differences, this method will still suffice.  

A microfluidic device would also contain the E.coli preventing them from entering the surrounding environment. Containment would be an obvious concern if a device like this were to ever hit the market. In this solution theoretically one could have pre-labeled E.coli in the machine stored and ready. The end user would simply intake a water sample right from the source, allow it to incubate, and process (sort) it. Collecting the larger fraction and viewing under a microscope would give a quick and easy real time method for detecting Dermo.

We believe that with the groundwork we have laid this year these future endeavors will be made possible as UMaryland continues to grow and gain expertise.