Difference between revisions of "Part:BBa K5415004"

Revision as of 08:17, 2 October 2024

To test the functionality of our biosensors, we first needed to “humanise” the yeast by introducing the HIF-1 system, as it was pivotal for mimicking hypoxic cellular reactions, akin to those experienced in human physiology, which, in turn, were crucial for our biosensor assays. By utilising the GAL1,10 promoter, we ensured robust expression of the HIF-1 components, as this promoter is known for its strong activation in the presence of galactose, thereby facilitating the desired gene expression under specific conditions. In our approach, we utilised two key iGEM BioBricks: HIF-1 beta and HIF-1 alpha, by integrating which we are able to create a functional hypoxia signalling cascade that closely resembles that found in human cells. ADH1 and ADH2 terminators were deployed to ensure precise regulation of gene expression and prevention of any unintended transcriptional read-through.

The successful implementation of this humanised yeast model opens up new avenues for biosensor development and testing. By leveraging the HIF-1 system, this composite part could not only contribute to a deeper understanding of cellular responses to oxygen deprivation but would also pave the way for innovative applications in medical diagnostics and/or environmental monitoring.

HIF–1α and HIF–1β expression

To further test the functionality of the HIF system in the humanised yeast strain, we aimed to test the expression of both HIF–1α and HIF–1β proteins. The humanised yeast strain contained the bidirectional GAL1,10 promoter, which is activated in the presence of galactose and the absence of glucose. Thus, we cultivated overnight cultures of our yeast strains in selective SD media containing 2% glucose, then washed the cells and cultivated them for 6 hours in SD media containing 0.5% glucose. After 6 hours we added galactose to the media to reach the final concentration of 2%. The yeast strains were then cultivated in these conditions for 20 hours. After 20 hours, cell samples were washed and fixed for immunofluorescence assay of both HIF–1α and HIF–1β proteins.

After incubation with primary (HIF–1 alpha Monoclonal Antibody or HIF–1 beta Monoclonal Antibody) antibodies, we used Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody conjugated with Alexa Fluor™ 488 to visualise the results.

The visualisation of results using epifluorescence microscopy (Alexa Fluor 488 filters, 1000x magnification) revealed interesting growth morphology of the yeast strains. In all samples, a varied count of elongated yeast cells were observed (Figures 1. and 2.). The elongated cells were emitting green light, not enough to be considered fluorescent, however, green colour, expected from Alexa Fluor, was present. The phenomenon of elongated cell morphology might be due to galactose induction, when no glucose and limited nitrogen were present in the media. In theory, this can lead to cell cycle arrest or abnormal cell morphology in yeast cells [1].

In comparison to immunofluorescence microscopy for HIF-1α, HIF–1β revealed more green light emission from the elongated cells. In figure 10, cells in both rectangles appear to be green, however, the yeast cell in rectangle 1 appears to be brighter.

In both immunofluorescence assays, green fluorescence was also observed. Figure 3 shows green fluorescence around the cells suggesting that the protein of interest is located outside of the cell. There is no reason to think that extracellular protein secretion has taken place as both proteins should be intracellular, however, the HIF system is not native to yeast cells and thus protein expression could differ from the HIF system in human cells.

Figure 4 shows fluorescence in yeast cells in HIF–1α immunofluorescence assay. The cell in the red rectangle shows fluorescence inside the cell. This confirms the HIF–1α protein expression in humanised yeast strain HG21.

One of the possible reasons for weak or no fluorescence observed in our humanised yeast immunofluorescence assays, could be the rapid degradation of HIF–1α in normal oxygen conditions, with a reported half-life of less than 5 minutes [2, 3]. The mechanism of HIF–1β degradation in normal oxygen conditions, however, is not known due to the fact that it is usually synthesised in hypoxic conditions only. To eliminate the rapid degradation of HIF system proteins to some extent, stabilisation techniques could be used and have to be tested.

References:

1. Ceccato-Antonini, S. R., & Sudbery, P. E. (2004). Filamentous growth in Saccharomyces cerevisiae. Brazilian Journal of Microbiology, 35(3), 173–181. https://doi.org/10.1590/s1517-83822004000200001
2. Marxsen, J. H., Stengel, P., Doege, K., Heikkinen, P., Jokilehto, T., Wagner, T., Jelkmann, W., Jaakkola, P., & Metzen, E. (2004). Hypoxia-inducible factor-1 (HIF-1) promotes its degradation by induction of HIF-α-prolyl-4-hydroxylases. Biochemical Journal, 381(3), 761–767. https://doi.org/10.1042/bj20040620
3. Hypoxia Inducible Factors (HIFS) | Bio-Techne. https://www.bio-techne.com/research-areas/hypoxia/hypoxia-inducible-factors-hif

HIF1 system for yeast humanisation.