Difference between revisions of "Part:BBa J63006:Experience"

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<I>British Columbia iGEM 2011</I>
 
<I>British Columbia iGEM 2011</I>
 
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Inconclusive results on our part since we have not yet sequenced but only used PCR to determine accurate placement of promoter and kozak sequence in front of GFP reporter. Please refer to characterization data below for more details.
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We have not yet sequenced but only used PCR to determine accurate placement of promoter and kozak sequence in front of GFP reporter. Please refer to characterization data below for more details. However, based on our results and analysis, we recommend using our <partinfo>BBa_K517000</partinfo> GAL promoter, which may be easier to connect to your construct since it lacks the ATG-containing Kozak Sequence present in the <partinfo>BBa_J63006</partinfo> GAL promoter. Again, please refer to our characterization for more details.
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|width='10%'|<partinfo>BBa_J63006 AddReview 4</partinfo>
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<I>Team:Shenzhen 2012</I>
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We have re-engineered this part to a new one BBa_K809605. We mutate the ATG at the 5' end to ACG, so it would not work as start codon. This could be useful for fusion protein construction and other purpose.
 
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<!-- DON'T DELETE --><partinfo>BBa_J63006 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_J63006 EndReviews</partinfo>
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==Characterization by Czech Republic iGEM 2015==
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The activity of pGAL1 promoter was characterized by transforming pGAL1-GFP plasmids into yeast S. cerevisiae and measuring the evolution of fluorescence in time.
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[[File:J63006Timeresponse.png|500px|left|border]]
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[[File:J63006Timeactivity.png|500px|left]]
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3 different concentrations (0.5%, 1%, 2%) of galactose were added to 100 µl yeast culture in SD min medium. We measured the fluorescence in 20 minutes intervals for 6 hours using a plate reader.
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The first graph shows evolution of fluorescence in time. We subtracted fluorescence of negative control from the fluorescence of each sample and divided this by difference between sample´s and negative control´s ODs in order to obtain RFU units.
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Our measurement showed that the higher concentrations causes quicker and higher activation of the promoter.
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Since 2%  appears to be the convenient concentration for maximal activation of the promoter in our experiment we used it as a reference and related the activity of other concentration to this concentration. Results are displayed in the second graph.
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The third graph show the static response characteristic, dependence of activation on concentration, related to the 2% concentration. 
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[[File:J63006_activity.png|450px|left]]
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==Characterization by British Columbia iGEM 2011==
 
==Characterization by British Columbia iGEM 2011==
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==Improvement by USTC 2016==
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Our part is Part:<html>
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<a href="https://parts.igem.org/Part:BBa_K2009363"> BBa_K2009363</a>
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</html>,designed by Kaiyue Ma. We construted this composite by ligating GAL1 promoter + Kozak sequence (Part:BBa_J63006) and (2) GFP (Part:BBa_E0040). However, the ATG inside the KOZAK sequence won't be in the same reading frame as the ATG of the downstream coding sequence, so a frame-shift mutation is inevitable. To solve this problem, we add a base pair after the Kozak sequence so that the ATG can be in the same reading frame and the GFP can be expressed properly. What’s more, this part become “ready to use”, which means the GFP sequence can be directly altered by other functional parts and the sequence will be expressed properly.
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==Improvement by ''' [http://2017.igem.org/Team:Kyoto  iGEM Kyoto 2017] ''' ==
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We used this promoter in order to express long hairpin RNA in yeast.
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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.
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The GPD promoter (BBa_K517001) was used as a control.
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For the data, please refer to the GPD Promoter page (''' [https://parts.igem.org/Part:BBa_K571001  BBa_K571001] ''') and '''[http://2017.igem.org/Team:Kyoto/Results our Wiki Result]'''.
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By feeding yeast expressing dsRNA to nematodes, we investigated whether the dsRNA expressed by these promoters is fatal to pine wood nematodes.

Latest revision as of 07:06, 1 November 2017

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_J63006

User Reviews

UNIQ40f12db0c2552e08-partinfo-00000000-QINU

•••

British Columbia iGEM 2011

We have not yet sequenced but only used PCR to determine accurate placement of promoter and kozak sequence in front of GFP reporter. Please refer to characterization data below for more details. However, based on our results and analysis, we recommend using our BBa_K517000 GAL promoter, which may be easier to connect to your construct since it lacks the ATG-containing Kozak Sequence present in the BBa_J63006 GAL promoter. Again, please refer to our characterization for more details.

••••

Team:Shenzhen 2012

We have re-engineered this part to a new one BBa_K809605. We mutate the ATG at the 5' end to ACG, so it would not work as start codon. This could be useful for fusion protein construction and other purpose.

UNIQ40f12db0c2552e08-partinfo-00000005-QINU

Characterization by Czech Republic iGEM 2015

The activity of pGAL1 promoter was characterized by transforming pGAL1-GFP plasmids into yeast S. cerevisiae and measuring the evolution of fluorescence in time.

J63006Timeresponse.png
J63006Timeactivity.png

3 different concentrations (0.5%, 1%, 2%) of galactose were added to 100 µl yeast culture in SD min medium. We measured the fluorescence in 20 minutes intervals for 6 hours using a plate reader.

The first graph shows evolution of fluorescence in time. We subtracted fluorescence of negative control from the fluorescence of each sample and divided this by difference between sample´s and negative control´s ODs in order to obtain RFU units.

Our measurement showed that the higher concentrations causes quicker and higher activation of the promoter.

Since 2% appears to be the convenient concentration for maximal activation of the promoter in our experiment we used it as a reference and related the activity of other concentration to this concentration. Results are displayed in the second graph.

The third graph show the static response characteristic, dependence of activation on concentration, related to the 2% concentration.


J63006 activity.png

Characterization by British Columbia iGEM 2011

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

The GPD promoter (BBa_K517001), GAL promoter (BBa_K517000) and GAL1 promoter with Kozak sequence (BBa_J63006) as characterized by their regulation of the expression of a GFP reporter.

Fluorescence Analysis of GFP expression as regulated by GPD and GAL Promoters: S. cerevisiae yeast strains containing either the GPD-GFP, GAL-GFP or GAL+Kozak-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 3 hours. The cells were spun down again and samples were collected at this 3 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 BBa_K517001 GPD promoter is constitutively activated and there is induction of the BBa_K517000 GAL promoter when it is shifted to galactose media. However, it is difficult to differentiate the expression of GFP under the regulation of the BBa_J63006 GAL Promoter with kozak sequence whether in glucose or galactose media. We have also analysed these by fluorescence activated cell sorting (FACS) (data available on BBa_K517000 and BBa_K517001 experience pages) but it remains ambiguous for the BBa_J63006 GAL Promoter. It would appear that were is a very weak constitutive expression of the GFP reporter under the BBa_J63006 GAL Promoter.

As a follow up to this experiment, we did a quick check of the BBa_J63006 GAL Promoter, but results were again not conclusive; there does not seem to be a clear induction of GFP expression when grown in galactose media.

Fluorescence Analysis of GFP expression as regulated by the GAL Promoters: S. cerevisiae yeast strains containing the GAL+Kozak-GFP construct were cultured at 30 degrees Celsius in either YPD (glucose) or SC-galactose media for 3 hours into log phase. The cells were 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.

DISCLAIMER: It may be that there was some error in cloning. We have to sequence to find out if we placed the BBa_J63006 GAL Promoter accurately into our GFP reporter construct. Nonetheless, PCR has been performed to confirm the presence of the BBa_J63006 GAL Promoter upstream of the GFP reporter. We are currently sequencing this construct and will have details at the Regional Jamboree 2011.

Another potential explanation for why this part may not have worked compared to our own BBa_K517000 GAL Promoter is that the kozak sequence may be interfering with expression of protein downstream; since the kozak sequence itself carries an ATG, it may be possible that this is competing with the ATG of the GFP to result in less expression even under galactose induction.

We aligned this (1) BBa_J63006 GAL Promoter+Kozak Sequence against our (2) BBa_K517000 GAL Promoter. There is 180bp upstream sequence present in (1) that is absent in (2), as well as the 27bp Kozak Sequence downstream of the identical sequence regions.



Improvement by USTC 2016


Our part is Part: BBa_K2009363 ,designed by Kaiyue Ma. We construted this composite by ligating GAL1 promoter + Kozak sequence (Part:BBa_J63006) and (2) GFP (Part:BBa_E0040). However, the ATG inside the KOZAK sequence won't be in the same reading frame as the ATG of the downstream coding sequence, so a frame-shift mutation is inevitable. To solve this problem, we add a base pair after the Kozak sequence so that the ATG can be in the same reading frame and the GFP can be expressed properly. What’s more, this part become “ready to use”, which means the GFP sequence can be directly altered by other functional parts and the sequence will be expressed properly.

Improvement 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 GPD promoter (BBa_K517001) was used as a control. For the data, please refer to the GPD Promoter page ( BBa_K571001 ) and [http://2017.igem.org/Team:Kyoto/Results our Wiki Result]. By feeding yeast expressing dsRNA to nematodes, we investigated whether the dsRNA expressed by these promoters is fatal to pine wood nematodes.