Engineering success
New Parts
As a result of the engineering design cycle, we have successfully developed the following parts that can detect oxygen.
In our project, we achieved engineering success by repeating many engineering design cycles, especially in the stage of realizing oxygen detection capability. The following steps illustrate our engineering success.
- Research and Design
- Build and Test
- Learn and Design
- Build and Test
Research and Design
We decided to focus on the oxygen-responsive genes inherent in yeast in order to implement the ability to detect oxygen in yeast. Yeast is well known for its Pasteur effect, and we thought that we could implement it smartly by using the pathway for this effect. Therefore, we first proceeded to research the oxygen-responsive gene cluster in yeast.
In the early stages of our research, we discussed which oxygen-responsive genes to focus on [4]. Since the protein HAP was found to play a major role in the oxygen response in yeast, further research on HAP revealed that CYC1 is regulated by Hap1 [1], we found that CYC1 is regulated by Hap1, and we also obtained the relationship between oxygen concentration and transcription level. The transcription level of CYC1 increased linearly around the threshold of oxygen concentration that we wanted to implement. Therefore, we thought that using the CYC1 promoter, we should not only be able to obtain oxygen responsiveness, but also be able to obtain the desired threshold with regulation.
Since we found that oxygen regulates the activity of Hap1 and Hap1 regulates the transcriptional level of CYC1, we decided to design a plasmid that utilizes this effect as follows. We thought that the strength of the fluorescence emitted by mCherry would be different depending on the oxygen concentration in yeast transfected with this plasmid.
Build and Test
We transformed the designed plasmid into yeast and tested whether the intensity of fluorescence changed in response to oxygen concentration. However, contrary to our expectation, there was no significant difference in fluorescence intensity between anaerobic and aerobic conditions, and mCherry fluorescence was observed in both conditions.
Learn and Design
Following the results of the first cycle of testing, we investigated why there was no difference in fluorescence intensity depending on oxygen concentration; transcriptional regulation by HAP involves changes in chromatin structure [2]. We hypothesized that since the CYC1 promoter was introduced into a plasmid, it could not be regulated by HAP and thus did not show oxygen responsiveness. This prediction is consistent with our experimental results showing that the CYC1 promoter in the plasmid worked as a constitutive promoter independent of oxygen.
Therefore, we decided to use a promoter that is not directly regulated by HAP, but instead is regulated by proteins that are coordinated by HAP. Specifically, we focused on ROX1, whose expression is promoted by HAP, and decided to use the ANB1 promoter and ROX1 promoter, which are regulated by ROX1, as oxygen-responsive promoters. These promoters have been found to work on plasmids in previous studies [3] [5], and we thought we could use them to confer oxygen responsiveness to yeast. We created a plasmid with mCherry attached downstream of these promoters.
Build and Test
The created plasmid was transformed into yeast and tested. The results showed that the fluorescence of the ANB1 promoter-transformed strain was stronger in aerobic conditions than in anaerobic conditions. This indicates that the ANB1 promoter is also oxygen-responsive in the plasmid, which brings us one step closer to one of the goals of our project, namely to create a device that detects and notifies changes in oxygen concentration when a wound is healing.
- 1.T. Hon et al., “A mechanism of oxygen sensing in yeast. Multiple oxygen-responsive steps in the heme biosynthetic pathway affect Hap1 activity,” J. Biol. Chem., vol. 278, no. 50, pp. 50771–50780, Dec. 2003. ↩
- 2.T. Zhang, P. Bu, J. Zeng, and A. Vancura, “Increased heme synthesis in yeast induces a metabolic switch from fermentation to respiration even under conditions of glucose repression,” J. Biol. Chem., vol. 292, no. 41, pp. 16942–16954, Oct. 2017. ↩
- 3.Z.-A. Fang et al., “Gene responses to oxygen availability in Kluyveromyces lactis: an insight on the evolution of the oxygen-responding system in yeast,” PLoS One, vol. 4, no. 10, p. e7561, Oct. 2009. ↩
- 4.K. E. Kwast, P. V. Burke, and R. O. Poyton, “Oxygen sensing and the transcriptional regulation of oxygen-responsive genes in yeast,” J. Exp. Biol., vol. 201, no. Pt 8, pp. 1177–1195, Apr. 1998. ↩
- 5.J. Deckert, A. M. Rodriguez Torres, J. T. Simon, and R. S. Zitomer, “Mutational analysis of Rox1, a DNA-bending repressor of hypoxic genes in Saccharomyces cerevisiae,” Mol. Cell. Biol., vol. 15, no. 11, pp. 6109–6117, Nov. 1995. ↩