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<p style="margin-left: 40px">Figure 4. Normalized fluorescence values produced under different promoters as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.</p> | <p style="margin-left: 40px">Figure 4. Normalized fluorescence values produced under different promoters as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.</p> | ||
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Latest revision as of 18:08, 20 October 2016
CCP1 promoter + Venus YFP
Introduction
CCP1 is a gene for cytochrome-c peroxidase. Cytochrome-c peroxidase is an enzyme that degrades reactive oxygen species in mitochondria. It is also involved in the response to cell’s oxidative stress. As Ccp1 is produced under oxidative stress, its promoter is activated in these conditions. (Saccharomyces genome database ID: S000001774)
We have created a device that uses oxidative stress as an inducer for reporter signal production by taking the promoter region (756 bp) from the CCP1 gene and fusing it to the protein-coding sequence of Venus yellow fluorescent protein. The length of promoter region is chosen to be the same previously used by He et al. (2005). With this length, the promoter contains all identified Skn7 and Yap1 binding sites and doesn’t overlap with the next gene in the genome. Skn7 and Yap1 are the main transcription factors activated in oxidative stress and are thus responsible for CCP1 promoter activation. The gene sequence for the CCP1 promoter region is obtained from strain S288C of Saccharomyces cerevisiae, from the Saccharomyces Genome Database.
The functionality of this promoter is tested in association with the Venus YFP reporter and proven to be functional when oxidative stress is induced by hydrogen peroxide.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 290
Illegal SpeI site found at 364 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 290
Illegal SpeI site found at 364 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 453
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 290
Illegal SpeI site found at 364 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 290
Illegal SpeI site found at 364
Illegal NgoMIV site found at 613
Illegal NgoMIV site found at 623 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 158
Illegal BsaI.rc site found at 1406
Previous use
We studied function of this device in the pRS415 plasmid backbone in S. cerevisiae strain SS328-leu. The device was inserted to the backbone so that it replaced multiple cloning site and the promoter site, and was directly followed by the cyc1 terminator.
Expression
Yeast containing the plasmids was precultured overnight at + 30 C with shaking in selective SD medium. The following morning, the cultures were refreshed with new medium to an OD of 0.2, and growth was continued until induction could be done at an OD of 0.5 by adding hydrogen peroxide. Hydrogen peroxide concentrations of 0-1mM allowed cell growth and YFP expression. A promoter expression profile was obtained by growing the induced cells in a microplate reader(Cytation3, BioTek) with vertical shaking at + 30 C; yellow fluorescence and cell density were measured at regular intervals. As the yeast easily formed clumps that interfere with the measurement, data over a longer time interval becomes inaccurate. The obtained expression profiles can be found in Figures 1 and 2. Because hydrogen peroxide has a negative effect on cell growth, plotting fluorescence as a function of cell density (OD600) gives more accurate information of whether the cells are producing more fluorescence when induced.
Figure 1. Yellow fluorescence produced under the CCP1 promoter as a function of time in different hydrogen peroxide concentrations.
Figure 2. Yellow fluorescence produced under the CCP1 promoter as a function of cell density (OD600) in different hydrogen peroxide concentrations.
These results were confirmed with flow cytometry analysis; cells were induced in the same way, and measured with a flow cytometer (FACSAria III) after 2 h and 4 h induction. The obtained fluorescence values from 2 hour induction are presented in figure 3.
Figure 3. Fluorescence values produced under different promoters as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.
Notably, the fluorescence seems to increase with hydrogen peroxide induction also in the control (GPD1 promoter). This could be due to the fact that because hydrogen peroxide slows culture growth, a relatively bigger portion of the cells in the culture are older, meaning that they have have accumulated more fluorescence. Because a defined number of cells are measured to obtain the fluorescence distribution, slower culture growth can show up as this kind of a bias. To account for this, fluorescence values were divided by the respective uninduced values of each promoter. The fluorescence values were normalized to uninduced values to gain an understanding of the resulting increase in fluorescence; the results for this are presented in figure 5.
Figure 4. Normalized fluorescence values produced under different promoters as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.
For more details on the experiments done with this BioBrick, visit http://2016.igem.org/Team:Aalto-Helsinki/Laboratory
References
He, X.J. and Fassler, J.S., 2005. Identification of novel Yap1p and Skn7p binding sites involved in the oxidative stress response of Saccharomyces cerevisiae. Molecular microbiology, 58(5), pp.1454-1467.