Difference between revisions of "Part:BBa K1907009"
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| − | 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. | + | 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 <i>Saccharomyces cerevisiae</i>, obtained from the Saccharomyces Genome Database. |
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<p style="margin-left: 40px">https://static.igem.org/mediawiki/parts/8/86/CTT1_1.png </p> | <p style="margin-left: 40px">https://static.igem.org/mediawiki/parts/8/86/CTT1_1.png </p> | ||
| − | <p style="margin-left: 40px">Figure 1. | + | <p style="margin-left: 40px">Figure 1. Cell density (OD600) and yellow fluorescence produced under the CTT1 promoter as a function of time in 0 mM hydrogen peroxide concentration.</p> |
<p style="margin-left: 40px">https://static.igem.org/mediawiki/parts/6/60/CTT1_2.png</p> | <p style="margin-left: 40px">https://static.igem.org/mediawiki/parts/6/60/CTT1_2.png</p> | ||
| − | <p style="margin-left: 40px">Figure 2. | + | <p style="margin-left: 40px">Figure 2. Cell density (OD600) and yellow fluorescence produced under the CTT1 promoter as a function of time in 1 mM hydrogen peroxide concentration. |
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| − | + | <b>References</b></p><p> | |
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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. | 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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Latest revision as of 00:56, 20 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
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. Cell density (OD600) and yellow fluorescence produced under the CTT1 promoter as a function of time in 0 mM hydrogen peroxide concentration.
Figure 2. Cell density (OD600) and yellow fluorescence produced under the CTT1 promoter as a function of time in 1 mM hydrogen peroxide concentration.
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.