Difference between revisions of "Part:BBa K517001"

Revision as of 02:39, 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

FACS 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 sonicated before running them through the FACS machine to determine the GFP expression profiles of each strain at each time point. Each profile was plotted with y-axis as counts of cells and x-axis as amount of GFP reading. The GPD-GFP strain's profile did not vary much over the course of the induction regardless of the media it was originally cultured in. However, the GAL-GFP strain experienced a comparably notable shift in the numbers of cells expressing higher levels of GFP when the strain was shifted into SC-galactose media. This profile shift was stronger when the strain was originally cultured in SC-raffinose instead of YPD (dextrose) media, which is expected since dextrose is known to more strongly repress the GAL promoter.

Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters

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.

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

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 )

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