Difference between revisions of "Part:BBa K1907009"
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<p style="margin-left: 40px">Figure 3. Fluorescence values as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence. | <p style="margin-left: 40px">Figure 3. Fluorescence values as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence. | ||
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The fluorescence values were normalized to uninduced values to gain an understanding of the relative increase in fluorescence; the results for this are presented in figure 4.</p> | The fluorescence values were normalized to uninduced values to gain an understanding of the relative increase in fluorescence; the results for this are presented in figure 4.</p> | ||
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<p style="margin-left: 40px">Figure 4. Normalized fluorescence values 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 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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| + | <p><b>References</b></p><p> | ||
| + | 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. <i>Molecular microbiology</i>, 58(5), pp.1454-1467. | ||
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Revision as of 20:50, 19 October 2016
CTT1 promoter + Venus YFP
Introduction
CTT1 is a gene for cytosolic catalase T. This protein has an important role in protecting the cell from oxidative damage caused by hydrogen peroxide. As Ctt1p is produced under oxidative stress, its promoter is activated in these conditions. (Saccharomyces genome database ID: S000003320)
We have created a device that uses oxidative stress as an inducer for reporter signal production by taking the promoter region (924 bp) from the CTT1 gene and fusing it to the protein-coding sequence of the Venus yellow fluorescent protein. The length of promoter region is chosen so that it contains identified Skn7 and Yap1 binding sites and doesn’t overlap with the next gene in the genome. (He et al., 2005) Skn7 and Yap1 are the main transcription factors activated in oxidative stress and are thus responsible for CTT1 promoter activation. In CTT1 promoter regions these binding sites are rather close to start codon (-147 bp), and as there are no genes close to it, a longer promoter are was decided to take just in case. The gene sequence for the CTT1 promoter region is taken from strain S288C of Saccharomyces cerevisiae, obtained from the Saccharomyces Genome Database.
The functionality of this promoter was tested in association with the Venus YFP reporter when oxidative stress was induced by hydrogen peroxide. Hydrogen peroxide didn’t seem to activate the promoter, but reaching stationary phase in cell growth did. This promoter could thus be used in monitoring the growth phase of yeast cells.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 402
Illegal PstI site found at 695 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 402
Illegal NheI site found at 701
Illegal PstI site found at 695 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 402
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 402
Illegal PstI site found at 695 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 402
Illegal PstI site found at 695
Illegal AgeI site found at 555 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 508
Illegal BsaI.rc site found at 1574
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, although increasing concentrations affect the absolute values of both negatively. 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 for two of the induction concentrations, along with corresponding OD600 profiles, can be found in Figures 1 and 2.
Figure 1. Yellow fluorescence produced under the TSA1 promoter as a function of time in different hydrogen peroxide concentrations.
Figure 2. Yellow fluorescence produced under the TSA1 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 as a function of different hydrogen peroxide concentrations. GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.
<p style="margin-left: 40px">
The fluorescence values were normalized to uninduced values to gain an understanding of the relative increase in fluorescence; the results for this are presented in figure 4.
Figure 4. Normalized fluorescence values as a function of different hydrogen peroxide concentrations.GPD1 is a negative control to assess background effects of hydrogen peroxide on fluorescence.
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.