Difference between revisions of "Part:BBa K1164008"

 
 
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__NOTOC__
 
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<partinfo>BBa_K1164008 short</partinfo>
 
<partinfo>BBa_K1164008 short</partinfo>
  
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This device consists of a modified version of the native Gal1 promoter found in S.cerevisiae driving production of yeast codon optimized GFP. The four gal4 binding domains present in the native Gal1 promoter have been replaced by four tetO sites. This promoter may be activated by regulatory activators that are able to bind to tetO. In addition, two lacO operators have been introduced downstream of the TATA box, allowing for repression of gene expression by lacI. Upon expression from the promoter, yEGFP will be produced.  
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This device consists of a modified version of the native Gal1 promoter found in S.cerevisiae driving production of yeast codon optimized GFP. The four gal4 binding domains present in the native Gal1 promoter have been replaced by four tetO sites. This promoter may be activated by regulatory activators that are able to bind to tetO. In addition, a lacO operator has been introduced downstream of the TATA box, allowing for repression of gene expression by lacI. Upon expression from the promoter, yEGFP will be produced.  
  
  
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<partinfo>BBa_K1164008 parameters</partinfo>
 
<partinfo>BBa_K1164008 parameters</partinfo>
 
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<br>
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https://static.igem.org/mediawiki/2013/6/6a/IPTGGraph2.png
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<p>The above data was collected from the following yeast strain: <br>
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<i>ade2</i>::Kanmx-rtact1-pgalLx-gfp<br>
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<i>ade4</i>::Natmx – rtact1-pgal-laci-bfp<br>
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In the above construct, pgallx drives the production of GFP and is repressed by Laci. As shown in the graph above, the repressor, Laci (tagged with BFP), remains relatively constant over the entire IPTG gradient: a chemical that represses the activity of Laci. Consequently, as IPTG concentration increases, Laci inhibition should be increasingly inhibited, resulting in more GFP being transcribed, which is supported by the above data.  The levels of fluorescence then stabilize and achieve steady state.</p>
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<"https://static.igem.org/mediawiki/2013/b/ba/PTRELX.png">
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<p>The above data was collected from the following yeast strain: <br>
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<br>
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ade2::Kan-rtACT1-pGalLX-rtTA-mCherry-VP16<br>
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ade4::Nat-rtACT1-pTRE-LacI-BFP
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<br>
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<br>
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This graphic shows that as the aTc concentration increases, PTRE is being increasingly activated, resulting in greater BFP transcription. This is expressed as the increase of fluorescence in the graphic above.
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<br>
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In the above construct, PTRE (driving BFP) is activated by rtTA , which is driven by pGallx and requires the presence of aTc. The levels of fluorescence then stabilize and achieve a steady level.
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</p>

Latest revision as of 20:56, 28 October 2013

pTRELX driving yeGFP with tPGK1 terminator

This device consists of a modified version of the native Gal1 promoter found in S.cerevisiae driving production of yeast codon optimized GFP. The four gal4 binding domains present in the native Gal1 promoter have been replaced by four tetO sites. This promoter may be activated by regulatory activators that are able to bind to tetO. In addition, a lacO operator has been introduced downstream of the TATA box, allowing for repression of gene expression by lacI. Upon expression from the promoter, yEGFP will be produced.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 465
    Illegal BamHI site found at 553
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 81
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1202



IPTGGraph2.png

The above data was collected from the following yeast strain:
ade2::Kanmx-rtact1-pgalLx-gfp
ade4::Natmx – rtact1-pgal-laci-bfp
In the above construct, pgallx drives the production of GFP and is repressed by Laci. As shown in the graph above, the repressor, Laci (tagged with BFP), remains relatively constant over the entire IPTG gradient: a chemical that represses the activity of Laci. Consequently, as IPTG concentration increases, Laci inhibition should be increasingly inhibited, resulting in more GFP being transcribed, which is supported by the above data. The levels of fluorescence then stabilize and achieve steady state.

<"PTRELX.png">

The above data was collected from the following yeast strain:

ade2::Kan-rtACT1-pGalLX-rtTA-mCherry-VP16
ade4::Nat-rtACT1-pTRE-LacI-BFP

This graphic shows that as the aTc concentration increases, PTRE is being increasingly activated, resulting in greater BFP transcription. This is expressed as the increase of fluorescence in the graphic above.
In the above construct, PTRE (driving BFP) is activated by rtTA , which is driven by pGallx and requires the presence of aTc. The levels of fluorescence then stabilize and achieve a steady level.