Difference between revisions of "Part:BBa J63006"
 
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===Contribution===
 
===Contribution===
  
−
<u>Addition of loop for hairpin-dsRNA to BBa_J63006</u>
+
<u>Addition of loop for hairpin-dsRNA to BBa_J63006</u><br>
 
''' [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_K2403005  BBa_K2403005] ''').
 
''' [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_K2403005  BBa_K2403005] ''').
  
Line 53: Line 53:
 
We attached a hairpin loop sequence behind this part so that you can easily make hairpin type dsRNA targeted to your favorite gene.
 
We attached a hairpin loop sequence behind this part so that you can easily make hairpin type 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.
 
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.
 +
 +
==Estonia_TUIT 2021 team contribution==
 +
===GAL1 promoter===
 +
 +
GAL1 promoter regulates the expression of the galactokinase gene. It is induced by galactose and repressed by glucose. The presence of galactose causes a 1000-fold increase in GAL1 gene transcription. Regulatory proteins Gal4 and Gal80 control the transcription. Without galactose, Gal80 inhibits GAL gene transcription. Galactose induction removes the Gal80 inhibition complex, allowing transcription activation by GAL4, which binds the upstream region of the GAL gene (Flick & Johnston, 1990).
 +
 +
===Plasmid construction===
 +
 +
To characterise the GAL1 promoter, we constructed plasmids using Golden Gate assembly and the MoClo yeast toolkit parts (Lee et al., 2015). Two plasmids were designed: low-copy number (CEN6/ARS origin of replication) and integrative plasmids (URA 3'-homology). We cloned the gene encoding for the Venus fluorescent reporter protein to these vectors under the control of our target promoter. The idea behind that is to detect, quantify and compare Venus fluorescence signals from plasmids with different copy numbers. The main features of the constructed plasmids are shown in Table 1.
 +
 +
'''''Table 1. The main features of constructed plasmids.'''''
 +
 +
<html>
 +
 +
  <head>
 +
      <title>HTML Tables</title>
 +
  </head>
 +
 +
  <body>
 +
      <table border = "1">
 +
        <tr>
 +
            <td>Promoter</td>
 +
            <td>Reporter</td>
 +
            <td>Origin of replication</td>
 +
            <td>Yeast integration site</td>
 +
            <td>Yeast selection gene</td>
 +
            <td>Copy number</td>
 +
        </tr>
 +
       
 +
        <tr>
 +
            <td>pGAL1</td>
 +
            <td>Venus</td>
 +
            <td>CEN6/ARS4</td>
 +
            <td>-</td>
 +
            <td>URA3</td>
 +
            <td>Low</td>
 +
        </tr>
 +
 +
        <tr>
 +
            <td>pGAL1</td>
 +
            <td>Venus</td>
 +
            <td>-</td>
 +
            <td>URA 3'-homology</td>
 +
            <td>URA3</td>
 +
            <td>Low</td>
 +
      </table>
 +
     
 +
  </body>
 +
</html>
 +
 +
===Yeast strain construction===
 +
 +
Constructed integration vectors were restricted with ''Not''I and used for transformation of ''S. cerevisiae'' DOM90 (MAT&alpha; ''{leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]'') strain. The CEN6/ARS4 plasmids were used without linearization. Transformants were selected for URA+ phenotype on uracil-dropout CSM plates containing 2% glucose. All yeast strains generated and used for promoter characterisation are listed in Table 2.
 +
 +
'''''Table 2. Yeast strains used for promoter characterisation.'''''
 +
 +
<html>
 +
 +
  <head>
 +
      <title>HTML Tables</title>
 +
  </head>
 +
 +
  <body>
 +
      <table border = "1">
 +
        <tr>
 +
            <td>Strain name</td>
 +
            <td>Genotype</td>
 +
            <td>Description</td>
 +
           
 +
        </tr>
 +
       
 +
        <tr>
 +
            <td>DOM90</td>
 +
            <td>MAT&alpha; <i>{leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]</i></td>
 +
            <td>Background strain without Venus (control) </td>
 +
           
 +
        </tr>
 +
 +
        <tr>
 +
            <td>ET53</td>
 +
            <td><i>pYTK074-CEN-pGAL1-Venus-tENO1-URA3</i></td>
 +
            <td>Strain with Venus under GAL1 promoter, low copy number</td>
 +
           
 +
        </tr>
 +
 +
        <tr>
 +
            <td>ET54</td>
 +
            <td><i>ura3-1::pYTK074-Integration-pGAL1-Venus-tENO1-URA3</i></td>
 +
            <td>Strain with Venus under GAL1 promoter, integration into <i>ura3-1</i> locus</td>
 +
      </table>
 +
     
 +
  </body>
 +
</html>
 +
 +
 +
===Microscopy===
 +
ET53, and ET54 strain cultures were grown in 3 ml of uracil-dropout CSM (2% raffinose) for 3 hours at 30˚C. After that, every strain culture was split into two: in one 2% galactose was added and the second culture was used as a no-induction control (with 2% glucose for GAL1 promoter repression). Cultures were grown for another 3 hours to OD<sub>600</sub> 0.2-0.8.
 +
 +
After that, 0.5 µl of cell culture was pipetted onto a 0.08 mm cover glass slip and covered with 1.5% agar-CSM (low melting temperature agarose was used). Zeiss Observer Z1 microscope with an automated stage, 63C/1.4NA oil immersion objective, and Axiocam 506 mono camera was used for imaging. During imaging, the focus was kept using Definite Focus and the sample was kept at 30°C using PeCon TempControl 37-2. ImageJ was used for image processing.
 +
 +
 +
===Results===
 +
We characterised the expression from the GAL1 promoter using Venus fluorescent protein as the reporter gene. The expression cassettes were either integrated to the yeast genome or on a centromeric low-copy plasmid, and the Venus expression was monitored in the presence and absence of the induction. GAL1 promoter was induced by addition of galactose. The experiments confirmed that the studied promoter is strongly regulated by the presence of the induction, as the Venus fluorescence intensity increased drastically upon induction (Figure 1). The expression from the chromosomal locus was lower than from plasmid. These experiments confirm that pGAL1 is a tightly-regulated promoter that mediates strong expression of the gene in the presence of the inductor.
 +
 +
<html>
 +
<center>
 +
<figure>
 +
                    <img style="width:100%;" src="https://2021.igem.org/wiki/images/6/67/T--Estonia_TUIT--Contribution_GAL1.jpg"><br><br>
 +
                    <i><b>Figure 1. Characterisation of pGAL1
 +
</b></I>Expression from GAL1 promoter is tightly controlled. The activity of the promoter was measured by quantifying the fluorescence intensity of Venus that is expressed from the promoter. The bars show mean fluorescence signals, error bars show standard deviation.
 +
                  </figure>
 +
</center>
 +
</html>
 +
 +
 +
===References===
 +
*Flick, J. S., & Johnston, M. (1990). Two Systems of Glucose Repression of the GAL] Promoter in Saccharomyces cerevisiae. MOLECULAR AND CELLULAR BIOLOGY, 10(9), 4757–4769.
 +
*Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975–986. https://doi.org/10.1021/SB500366V

Latest revision as of 09:24, 21 October 2021

yeast GAL1 promoter

GAL1 promoter from S. cerevisiae Contains Kozak sequence, part BBa_J63003.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 150
  • 1000
    COMPATIBLE WITH RFC[1000]


Promoter characterization

Team Czech Republic 2015 used this promoter very extensively. As a result, the characterization of this part was improved. See the Experience page for details.

Promoter Strength

JHU Igem 2011 is working on further characterization of this part through using GFP expression within the next month.


Promoter improvement

Team USTC 2016 modify this part to make it "ready to use". It is more convenient to be utilized now. See the Experience page for details.

Promoter dose =

Gal1 prevents growth in media containing galactose.

SCUT iGEM 2014 have designed a experiment to quantify Gal1 dose titer.


Figure 1.This year we have .



















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. GPD promoter ( BBa_K571001 ) 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].


Contribution

Addition of loop for hairpin-dsRNA to BBa_J63006
[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_K2403005 ).

In order to kill pine wood nematodes by feeding RNAi, we used this part as a promoter to express hairpin-loop 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 behind this part so that you can easily make hairpin type 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.

Estonia_TUIT 2021 team contribution

GAL1 promoter

GAL1 promoter regulates the expression of the galactokinase gene. It is induced by galactose and repressed by glucose. The presence of galactose causes a 1000-fold increase in GAL1 gene transcription. Regulatory proteins Gal4 and Gal80 control the transcription. Without galactose, Gal80 inhibits GAL gene transcription. Galactose induction removes the Gal80 inhibition complex, allowing transcription activation by GAL4, which binds the upstream region of the GAL gene (Flick & Johnston, 1990).

Plasmid construction

To characterise the GAL1 promoter, we constructed plasmids using Golden Gate assembly and the MoClo yeast toolkit parts (Lee et al., 2015). Two plasmids were designed: low-copy number (CEN6/ARS origin of replication) and integrative plasmids (URA 3'-homology). We cloned the gene encoding for the Venus fluorescent reporter protein to these vectors under the control of our target promoter. The idea behind that is to detect, quantify and compare Venus fluorescence signals from plasmids with different copy numbers. The main features of the constructed plasmids are shown in Table 1.

Table 1. The main features of constructed plasmids.

HTML Tables

Promoter Reporter Origin of replication Yeast integration site Yeast selection gene Copy number
pGAL1 Venus CEN6/ARS4 - URA3 Low
pGAL1 Venus - URA 3'-homology URA3 Low

Yeast strain construction

Constructed integration vectors were restricted with NotI and used for transformation of S. cerevisiae DOM90 (MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+]) strain. The CEN6/ARS4 plasmids were used without linearization. Transformants were selected for URA+ phenotype on uracil-dropout CSM plates containing 2% glucose. All yeast strains generated and used for promoter characterisation are listed in Table 2.

Table 2. Yeast strains used for promoter characterisation.

HTML Tables

Strain name Genotype Description
DOM90 MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15 bar1::hisG} [phi+] Background strain without Venus (control)
ET53 pYTK074-CEN-pGAL1-Venus-tENO1-URA3 Strain with Venus under GAL1 promoter, low copy number
ET54 ura3-1::pYTK074-Integration-pGAL1-Venus-tENO1-URA3 Strain with Venus under GAL1 promoter, integration into ura3-1 locus


Microscopy

ET53, and ET54 strain cultures were grown in 3 ml of uracil-dropout CSM (2% raffinose) for 3 hours at 30˚C. After that, every strain culture was split into two: in one 2% galactose was added and the second culture was used as a no-induction control (with 2% glucose for GAL1 promoter repression). Cultures were grown for another 3 hours to OD600 0.2-0.8.

After that, 0.5 µl of cell culture was pipetted onto a 0.08 mm cover glass slip and covered with 1.5% agar-CSM (low melting temperature agarose was used). Zeiss Observer Z1 microscope with an automated stage, 63C/1.4NA oil immersion objective, and Axiocam 506 mono camera was used for imaging. During imaging, the focus was kept using Definite Focus and the sample was kept at 30°C using PeCon TempControl 37-2. ImageJ was used for image processing.


Results

We characterised the expression from the GAL1 promoter using Venus fluorescent protein as the reporter gene. The expression cassettes were either integrated to the yeast genome or on a centromeric low-copy plasmid, and the Venus expression was monitored in the presence and absence of the induction. GAL1 promoter was induced by addition of galactose. The experiments confirmed that the studied promoter is strongly regulated by the presence of the induction, as the Venus fluorescence intensity increased drastically upon induction (Figure 1). The expression from the chromosomal locus was lower than from plasmid. These experiments confirm that pGAL1 is a tightly-regulated promoter that mediates strong expression of the gene in the presence of the inductor.



Figure 1. Characterisation of pGAL1 Expression from GAL1 promoter is tightly controlled. The activity of the promoter was measured by quantifying the fluorescence intensity of Venus that is expressed from the promoter. The bars show mean fluorescence signals, error bars show standard deviation.


References

  • Flick, J. S., & Johnston, M. (1990). Two Systems of Glucose Repression of the GAL] Promoter in Saccharomyces cerevisiae. MOLECULAR AND CELLULAR BIOLOGY, 10(9), 4757–4769.
  • Lee, M. E., DeLoache, W. C., Cervantes, B., & Dueber, J. E. (2015). A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly. ACS Synthetic Biology, 4(9), 975–986. https://doi.org/10.1021/SB500366V