Difference between revisions of "Part:BBa K517001"
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===Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters=== | ===Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters=== | ||
[[Image:Ubcigem2011Promoterimages0.jpg | frame | center | '''Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters:''' ''S. cerevisiae'' yeast strains containing either the GPD-GFP or GAL-GFP construct were cultured overnight at 30 degrees Celsius in either YPD (dextrose), SC-raffinose or SC-galactose media. These were diluted 1 in 10 in their respective media and grown for 3 hours at 30 degrees Celsius into log phase. The cells were then spun down and samples were collected at 0 hours. The remaining cells were resuspended in SC-galactose media and left to grow at 30 degrees Celsius for 2 hours. The cells were spun down again and samples were collected that this 2 hour time point. The samples were fixed in paraformaldehyde and visualized under a fluorescence microscope under the GFP and DIC settings. Acquired images were then color-combined with red representing DIC and green representing GFP. Microscopy images show that the GPD promoter results in constitutive high expression while the GAL promoter is induced by the shift to galactose media.]] | [[Image:Ubcigem2011Promoterimages0.jpg | frame | center | '''Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters:''' ''S. cerevisiae'' yeast strains containing either the GPD-GFP or GAL-GFP construct were cultured overnight at 30 degrees Celsius in either YPD (dextrose), SC-raffinose or SC-galactose media. These were diluted 1 in 10 in their respective media and grown for 3 hours at 30 degrees Celsius into log phase. The cells were then spun down and samples were collected at 0 hours. The remaining cells were resuspended in SC-galactose media and left to grow at 30 degrees Celsius for 2 hours. The cells were spun down again and samples were collected that this 2 hour time point. The samples were fixed in paraformaldehyde and visualized under a fluorescence microscope under the GFP and DIC settings. Acquired images were then color-combined with red representing DIC and green representing GFP. Microscopy images show that the GPD promoter results in constitutive high expression while the GAL promoter is induced by the shift to galactose media.]] | ||
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+ | ==Characterization by ''' [http://2017.igem.org/Team:Kyoto iGEM Kyoto 2017] ''' == | ||
+ | We used this promoter in order to express long hairpin RNA in yeast. | ||
+ | Long hairpin RNA targeting <i>B. xylophilus</i> AK1 mRNA or GFP mRNA was cloned downstream of the promoter and introduced into the 2-micron high copy number plasmid of budding yeast, and subsequently expressed in yeast. | ||
+ | The Gal1 promoter (''' [https://parts.igem.org/Part:BBa_J63006 BBa_J63006] ''') was used as a control. | ||
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+ | Results of detection by qRT-PCR were as follows | ||
+ | <!-- 以下の表に相当するfigを | ||
+ | Medium Stock plasmid Average value SD | ||
+ | Gal MKY13 WT Gal1p-AK1 3.61 0.98 | ||
+ | Glu MKY13 WT Gal1p-AK1 0.11 0.00 | ||
+ | Glu MKY13 WT GPD-AK1 0.07 0.02 | ||
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+ | (qRT-PCR targeted to the loop portion of hairpin. Data was normalized with 25S rRNA. n=3 ) | ||
+ | --> | ||
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+ | [[File:プロモーター比較表.jpg|500px|thumb|center|'''Figure 1 : Results of detection by qRT-PCR (qRT-PCR targeted to the loop portion of hairpin. Data was normalized with 25S rRNA. n=3 )''']] | ||
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+ | As a result, it was revealed that the constitutive GPD promoter had low RNA expression level. That level was even lower than from the conditional Gal1 promoter (BBa_J63006) in Glucose (Glu) medium, a condition which suppresses expression from Gal1promoter. We conducted another experiment with a longer 500 bp promoter which contains this GPD promoter sequence(we used TDH3 promoter of ''' [https://parts.igem.org/Part:BBa_K530008 BBa_K530008] '''), and it was confirmed that the longer promoter has stronger expression. Therefore we cannot recommend using this part (BBa_K517001) for expressing protein excessively. | ||
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+ | ==Contribution== | ||
+ | ===Addition of loop for hairpin-dsRNA to BBa_K517001=== | ||
+ | ''' [http://2017.igem.org/Team:Kyoto iGEM Kyoto 2017] ''' has modified this part so that it can also be used for dsRNA expression (see ''' [https://parts.igem.org/Part:BBa_K2403004 BBa_K2403004] '''). | ||
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+ | In order to kill pine wood nematodes by feeding RNAi, we used this part as a promoter to express hairpin-dsRNA in yeast. Then we confirmed the expression level of dsRNA in yeast by qRT-PCR and fed the yeast to pine wood nematodes. | ||
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+ | We attached a hairpin loop sequence to this part so that you can easily make hairpin loop- dsRNA targeted to your favorite gene. | ||
+ | You can create a plasmid transcribing dsRNA with a hairpin loop which can be expressed in budding yeast by cleaving with the restriction enzyme NotI, connecting the sense part between the promoter and loop parts, then cleaving with the restriction enzyme HindIII for ligating the antisense part. | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here |
Latest revision as of 02:40, 2 November 2017
GPD constitutive yeast promoter
Promoter for constitutively high expression.
Reference: This promoter is commonly used in several yeast vectors including the [http://www.addgene.org/yeast-gateway/ Advanced Gateway Vectors].
Book reference: Christine Guthrie, Gerald R. Fink. 2004. Guide to Yeast Genetics and Molecular and Cell Biology, Volume 1. Gulf Professional Publishing. Chapter 26: Vectors for Constitutive and Inducible Gene Expression in Yeast. Page 391.
Characterization by British Columbia iGEM 2011
The GPD promoter (BBa_K517001) and GAL promoter (BBa_K517000) as characterized by their regulation of the expression of a GFP reporter.
FACS Analysis of GFP expression as regulated by GPD and GAL Promoters
Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters
Characterization by [http://2017.igem.org/Team:Kyoto iGEM Kyoto 2017]
We used this promoter in order to express long hairpin RNA in yeast. Long hairpin RNA targeting B. xylophilus AK1 mRNA or GFP mRNA was cloned downstream of the promoter and introduced into the 2-micron high copy number plasmid of budding yeast, and subsequently expressed in yeast. The Gal1 promoter ( BBa_J63006 ) was used as a control.
Results of detection by qRT-PCR were as follows
As a result, it was revealed that the constitutive GPD promoter had low RNA expression level. That level was even lower than from the conditional Gal1 promoter (BBa_J63006) in Glucose (Glu) medium, a condition which suppresses expression from Gal1promoter. We conducted another experiment with a longer 500 bp promoter which contains this GPD promoter sequence(we used TDH3 promoter of BBa_K530008 ), and it was confirmed that the longer promoter has stronger expression. Therefore we cannot recommend using this part (BBa_K517001) for expressing protein excessively.
Contribution
Addition of loop for hairpin-dsRNA to BBa_K517001
[http://2017.igem.org/Team:Kyoto iGEM Kyoto 2017] has modified this part so that it can also be used for dsRNA expression (see BBa_K2403004 ).
In order to kill pine wood nematodes by feeding RNAi, we used this part as a promoter to express hairpin-dsRNA in yeast. Then we confirmed the expression level of dsRNA in yeast by qRT-PCR and fed the yeast to pine wood nematodes.
We attached a hairpin loop sequence to this part so that you can easily make hairpin loop- dsRNA targeted to your favorite gene. You can create a plasmid transcribing dsRNA with a hairpin loop which can be expressed in budding yeast by cleaving with the restriction enzyme NotI, connecting the sense part between the promoter and loop parts, then cleaving with the restriction enzyme HindIII for ligating the antisense part.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]