Our project has three main areas of focus:
1) Design and build low-cost open source biotools:
Our interdisciplinary backgrounds and our convenient home within Inworks at the University of Colorado
Denver, allowed our team to build many of the tools we use in the lab including a fluorimeter, an optical density sensor,
a DNA concentration meter, an incubator, a refrigerated centrifuge, and a yeast dryer,
which we will be exhibiting at the Jamboree expo as part of our "Hardware" track deliverables.
The fluorimeter and optical density sensor we built was based on a design by the 2014 Aachen iGEM team. We improved upon the original design by designing a 3-in-1 cuvette holder using a Form 2 Resin 3D printer. Different colored filters can be inserted interchangeably into the cuvette holder to filter different frequencies of light, and thus detect opitcal density or fluoresence or DNA concentration. We also improved upon the original Arduino code by refactoring and commenting functions. We also used a four-button Olimex LCD screen, which required re-mapping functions to the four different buttons, and adding DNA concentration calculations to the code base. All of our code is open source and available for download on our github repository.
The high-level goal of our project is make biological diagnostics and tools more accessible in resource-constrained areas. The difficulty with implementing biological diagnostic tools in these places, however, is largely the same as the issues that orginally motivated our project: lack of refrigeration and resources. We therefore wanted to design a system where engineered yeast could be "dried" similar to the process for bakers and brewers yeast, shipped in inexpensive packets at room temperature, and reconstituted on site to preform the intended diagnostic funtion. To meet this goal we built a yeast drier, modeled after industrial yeast driers. Our Project Design and Proof of Concept pages contain more details on the construction and implementation of our hardware designs.
2) Create an oxytocin quality sensor using a G-protein coupled receptor in yeast:
The second goal of our project was to design and test a diagnostic system for medication quality
that leveraged G-protein coupled receptors (GPCRs) in yeast. We relied on the DIY receptor design instructions created by
TU-Delft's 2012 iGEM team to create our own
oxytocin GPCR. TU-Delft's design leveraged work done by Radhika et. al.
who engineered Saccharomyces cerevisiae to contain components of the mammalian (rat) olfactory signaling
pathway to detect dinitrotoluene, an explosive residue. TU-Delft used this design to create
Snifferomyces, a yeast strain capable of detecting Niacin, a compound exhaled by individuals
with tuberculosis. Our goal was to use the same GPCR approach to design yeast that could detect
active oxytocin in order to determine if the medication sample was expired or not.
We began by designing our receptor using SnapGene (one of our generous sponsors) and having it synthesized by IDT (for free as part of their sponsorship of iGEM 2016, thank you!) We then cloned our OXTR part into the TU-Delft part from the BioBrick distribution kit to create a composite part. We used 3A assembly to remove the composite part which included a yeast promoter, a RBS, our OXTR part, a terminator, and the FUS-1 and EGFP parts from TU-Delft.
Our next step was to order several shuttle vectors from AddGene so we could produce more of our part using E.coli, mini-prep it out, and transform it into yeast where it would express the proteins required for our oxytocin receptor to form on the cell's surface, and when oxytocin is detected, trigger the FUS-1 mating pathway and produce measureable green fluorescent protein.
In our work on this aspect of the project we experimented with different reporters, yeast promoters and terminators, yeast strains, and selection techniques. More details can be found on our Project Design and Proof of Concept pages.
3) Produce a more temperature stable form of oxytocin using E.coli and/or yeast:
The third, and original goal, of our project was to directly address the
short half-life and need for refrigeration of the life-saving drug oxytocin. We have begun work on this
aspect of the project in several ways. First, we researched E.coli strains capable of folding human proteins,
and ordered BL21. We also began testing different competent cell protocols to maximize our transformation
efficiency of this strain. We also requested a part iGEM HQ
submitted by the Lethbridge Canada 2013 iGEM team .
The Lethbridge part included the oxytocin gene and its precursor molecule nueorphysin-I. The goal of the Lethbridge
team was also to create a longer-lasting form of oxytocin by adding precursor molecules to slow the hormone's degradation.
We are working to improve upon their part by experimenting with different secretion tags to facilitate easier
precipitation of the resulting protein.
We are still working on this aspect of the project, researching possible mechanisms for producing the human oxytocin protein using yeast, and exploring possible temperature regulation mechanisms to mitigate the instability of this essential medication.