Difference between revisions of "Part:BBa K1907008"

 
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<p><b>Introduction</b></p><p>
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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)
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</p><p>
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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 (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.
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</p><p>
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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.</p>
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<span class='h3bb'><p><b>Sequence and Features</b></p><p>
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<partinfo>BBa_K1907008 SequenceAndFeatures</partinfo>
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</p><p>
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</p><p>
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<p><b>Previous use</b></p><p>
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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.
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<p style="margin-left: 40px"><b>Expression</b></p>
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<p style="margin-left: 40px">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.</p>
  
 
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Revision as of 20:17, 19 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 290
    Illegal SpeI site found at 364
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 290
    Illegal SpeI site found at 364
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 453
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 290
    Illegal SpeI site found at 364
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 158
    Illegal BsaI.rc site found at 1406

<p>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.

<p style="margin-left: 40px">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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 290
    Illegal SpeI site found at 364
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 290
    Illegal SpeI site found at 364
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 453
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 290
    Illegal SpeI site found at 364
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 158
    Illegal BsaI.rc site found at 1406