Vision for the future
Things to improve
There are two construct stages and one freeze-drying process in our project, among these stages we have made promising achievements, mainly in demonstrating our yeast glucose and epinephrine sensor can function in lab and real-world conditions.
However, there are two main problems left unsolved:
Problem 1:The large variations between different experimental groups
Solution: More parallel groups.As shown in previous pages, although some of our results displayed a statistical significance with P values smaller than 0.05, the variations between groups are still quite large. Luminescent signals were relatively unstable when compared with fluorescent signal, especially those based on alternation in enzyme activity due to conformational changes. Thus, in order to get a more reliable result, we need more parallel groups to minimize the standard deviation and also, to further demonstrate our device.
Problem 2:The final connection of our product to a digital platform to create a yeast testing kit.
Solution: Smartly designed testing kit.Professor Nagai demonstrate the fusion protein, Nano-lantern, possess the ability to emit lights which can be observed using bare eyes, and its sibling, Nano-lantern (cAMP 1.6), is 0.2~0.4 to this value [1]. In our experiments, we indeed observed the luminescence with our bare eyes, while we failed to use our cell phone to record that because the phone camera is hard to focus in dark conditions. So we need to modify our testing sets with engineering design, we need a black box to put our biosensor and pathological samples as well as the camera into it to make measurements.
Things to expand - The GPCR collection
The versatility of our yeast chassis comes from the wide range of a patterned molecules, G-protein coupled receptors. So far, numerous GPCRs have been expressed in yeast Saccharomyces cerevisiae, including human adenosine receptor A1, human dopamine receptor D2L, human neurotensin receptor type-1 and human UDP-glucose receptor[2, 3]. So far, most difficulties in heterologous expression of functional exogenous GPCR in yeast are mainly due to membrane localization and functional coupling with yeast endogenous G proteins. The N terminus of yeast endogenous GPCR and previously successfully expressed exogenous GPCR can all be used to increase the ability to be localized on membrane [4, 5]. Chimeric G proteins have been proposed as a solution to low signal coupling between molecules, and the C terminus of a successfully expressed GPCR can also serve as an alternate [4, 6]. By means of genetic engineering, we firmly believe that the range of detectable molecule could be largely widened using our existing yeast chassis.
Reference
[1] Saito, K., et al., Luminescent proteins for high-speed single-cell and whole-body imaging. Nat Commun, 2012. 3: p. 1262.
[2] O'Malley, M.A., et al., Progress toward heterologous expression of active G-protein-coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking. Protein Science, 2009. 18(11): p. 2356-2370.
[3] Ishii, J., et al., Microbial fluorescence sensing for human neurotensin receptor type 1 using G alpha-engineered yeast cells. Analytical Biochemistry, 2014. 446: p. 37-43.
[4] Radhika, V., et al., Chemical sensing of DNT by engineered olfactory yeast strain. Nature Chemical Biology, 2007. 3(6): p. 325-330.
[5] King, K., et al., Control of yeast mating signal transduction by a mammalian beta 2-adrenergic receptor and Gs alpha subunit. Science, 1990. 250(4977): p. 121-3.
[6] Ishii, J., et al., Improved identification of agonist-mediated G alpha(i)-specific human G-protein-coupled receptor signaling in yeast cells by flow cytometry. Analytical Biochemistry, 2012. 426(2): p. 129-133.
