Team:Rice University/CONSTRUCTS

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[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] :::  [[Team:Rice_University/BACKGROUND|BACKGROUND]] :::  [[Team:Rice_University/STRATEGY|STRATEGY]] :::   [[Team:Rice_University/RESULTS|RESULTS]] :::  [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]] ::: 
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[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] :::  [[Team:Rice_University/BACKGROUND|BACKGROUND]] :::   
 +
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] :::  [[Team:Rice_University/RESULTS|RESULTS]] :::  [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]
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==='''Constructs'''===
==='''Constructs'''===
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     <p>''Saccharomyces cerevisiae'' are widely used for baking and brewing, and they are particular useful for synthesizing metabolites under fermentation conditions which prevent the air oxidation of many useful compounds. To achieve our project and expand the synthetic biology toolbox for programming yeast, we have introduced into the iGem registry BioBricks encoding 3 yeast promoters, 3 yeast terminators, a two micron origin of replication, 2 selectable markers, 2 enzymes, and a yeast integration plasmid. In addition, we have generated seven constructs using these parts.  Furthermore, we have submitted two additional parts representing a foundational tool, including a gene encoding an amber suppressed RFP biobrick for screening of SupF+ (Amber suppressor) genotype and an amber suppressor tRNA biobrick. <br />
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     <p>''Saccharomyces cerevisiae'' is widely used for baking and brewing, a versatile eukaryotic model system,  and particularly useful for synthesizing metabolites under fermentation conditions. The microaerobic conditions of fermentation impede the oxidation of sensitive bioreactive compounds and are optimal for the ''de novo'' synthesis of resveratrol. To achieve our project goals and expand the yeast synthetic biology toolbox, we have constructed BioBricks encoding 3 yeast promoters, 3 yeast terminators, a 2-micron origin of replication, 2 selectable markers, 2 metabolic enzymes, and a yeast integration plasmid. We have also submitted two additional parts representing foundational tools, including a gene encoding an amber suppressed RFP biobrick for screening of SupF+ (Amber suppressor) genotype and an amber suppressor tRNA biobrick. <br />
     </p>
     </p>
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         <td width="120">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 BBa_K122000]</td>
         <td width="120">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 BBa_K122000]</td>
         <td width="118">pPGK1</td>
         <td width="118">pPGK1</td>
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         <td width="466">This is the 1500 bp upstream of the PGK1 coding region in an industrial yeast strain. Constitutive promoter. </td>
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         <td width="466">~1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation. </td>
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         <td width="24">1497</td>
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         <td width="24">1497bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122002 BBa_K122002]</td>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122002 BBa_K122002]</td>
         <td>pADH1</td>
         <td>pADH1</td>
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         <td>700bp upstream of ADH1 promoter region containing RBS. Constitutive promoter.</td>
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         <td>700bp upstream of ADH1 promoter region. Constitutive promoter under aerobic and anaerobic conditions.</td>
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         <td>701</td>
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         <td>701bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122017]</td>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122017]</td>
         <td>pGAL1 + tetO</td>
         <td>pGAL1 + tetO</td>
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         <td>The GAL1 promoter which is highly repressed by glucose. An additional tetracycline operator site was included upstream of the RBS to allow repression by tetR.</td>
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         <td>Glucose repressible / galactose inducible GAL1 promoter. An additional TetR operator site was included to allow repression by TetR.</td>
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         <td>484</td>
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         <td>484bp</td>
       </tr>
       </tr>
     </table>
     </table>
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         <td width="74">tCYC1</td>
         <td width="74">tCYC1</td>
         <td width="493">300bp downstream the CYC1 coding region in a standard yeast strain.</td>
         <td width="493">300bp downstream the CYC1 coding region in a standard yeast strain.</td>
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         <td width="46">300</td>
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         <td width="46">300bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>tADH1</td>
         <td>tADH1</td>
         <td>300bp downstream the ADH1 coding region in a standard yeast strain.</td>
         <td>300bp downstream the ADH1 coding region in a standard yeast strain.</td>
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         <td>300</td>
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         <td>300bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>tPGK1</td>
         <td>tPGK1</td>
         <td>1000bp downstream the PGK1 coding region in an industrial yeast strain.</td>
         <td>1000bp downstream the PGK1 coding region in an industrial yeast strain.</td>
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         <td>1000</td>
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         <td>1000bp</td>
       </tr>
       </tr>
     </table>
     </table>
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         <td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122018]</td>
         <td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122018]</td>
         <td width="74">ZeoR</td>
         <td width="74">ZeoR</td>
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         <td width="493">Zeocin Resistance Gene</td>
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         <td width="493">Zeocin/Bleocin Resistance Gene</td>
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         <td width="46">300</td>
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         <td width="46">300bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>BleoR</td>
         <td>BleoR</td>
         <td>Bleocin Resistance Gene under pTet promoter</td>
         <td>Bleocin Resistance Gene under pTet promoter</td>
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         <td>800ish</td>
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         <td>80bp-ish</td>
       </tr>
       </tr>
       <tr>
       <tr>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122014 BBa_K122014]</td>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122014 BBa_K122014]</td>
         <td>ORI+HisTag</td>
         <td>ORI+HisTag</td>
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         <td>2 Micron ORI and Histadine Tag </td>
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         <td>2 Micron ORI and auxotrophic histidine marker </td>
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         <td>&lt;9000</td>
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         <td>&lt;9000bp</td>
       </tr>
       </tr>
     </table>
     </table>
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         <td width="451">[pGAL1][tetO][ZeoR]</td>
         <td width="451">[pGAL1][tetO][ZeoR]</td>
         <td width="74">[[Image:C2.jpg|120px]]</td>
         <td width="74">[[Image:C2.jpg|120px]]</td>
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         <td width="90">&nbsp;</td>
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         <td width="90">874bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>Tyrosine Ammonia Lyase</td>
         <td>Tyrosine Ammonia Lyase</td>
         <td>[[Image:TAL.jpg|50px]]</td>
         <td>[[Image:TAL.jpg|50px]]</td>
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         <td>&nbsp;</td>
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         <td>1933bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]</td>
         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]</td>
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         <td>4CL:STS</td>
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         <td>4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein</td>
         <td>[[Image:4CL.jpg|50px]]</td>
         <td>[[Image:4CL.jpg|50px]]</td>
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         <td>&nbsp;</td>
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         <td>4000bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>[pPGK1][4CL:STS][tCYC1]</td>
         <td>[pPGK1][4CL:STS][tCYC1]</td>
         <td>[[Image:C1.jpg|150px]]</td>
         <td>[[Image:C1.jpg|150px]]</td>
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         <td>&nbsp;</td>
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         <td>5497bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>[pGAL1][tetO][ZeoR][tADH1]</td>
         <td>[pGAL1][tetO][ZeoR][tADH1]</td>
         <td>[[Image:C2full.jpg|150px]]</td>
         <td>[[Image:C2full.jpg|150px]]</td>
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         <td>&nbsp;</td>
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         <td>1175bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>[pADH1][TAL][tPGK1]</td>
         <td>[pADH1][TAL][tPGK1]</td>
         <td>[[Image:C3.jpg|150px]]</td>
         <td>[[Image:C3.jpg|150px]]</td>
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         <td>&nbsp;</td>
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         <td>2651bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
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         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122016 BBa_K122019]</td>
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         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122019 BBa_K122019]</td>
         <td>[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1]</td>
         <td>[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1]</td>
         <td>[[Image:C1C2C3.jpg|350px]]</td>
         <td>[[Image:C1C2C3.jpg|350px]]</td>
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         <td>&nbsp;</td>
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         <td>1824bp</td>
       </tr>
       </tr>
     </table>
     </table>
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Novel Zero Leak Inverter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006])
Novel Zero Leak Inverter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006])
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[[Image:tRNA.png|thumb|250px|left|tRNA]]
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[[Image:tRNA.png|thumb|250px|left|Amber Suppressor tRNA]]
<table width="477" border="1">
<table width="477" border="1">
       <tr>
       <tr>
         <td width="71">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007]</td>
         <td width="71">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007]</td>
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         <td width="354">The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons.</td>
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         <td width="354">The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons.  Charges with tyrosine.</td>
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         <td width="30">211</td>
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         <td width="30">211bp</td>
       </tr>
       </tr>
       <tr>
       <tr>
         <td height="54">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006]</td>
         <td height="54">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006]</td>
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         <td>Point Mutation of RFP(13521) with incorporation of an amber stop codon at the chemophore.</td>
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         <td>Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation.</td>
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         <td>923</td>
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         <td>923bp</td>
       </tr>
       </tr>
     </table>
     </table>
 +
 +
<BR><BR>
      
      
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Through incorporation of amber stop codons or point mutations of tyrosine codons (TAC) to TAG within the coding region, a genetic circuit can be used to add an additional level of regulation and determine whether a full protein or partial peptide with be synthesized. This design has high signal-to-noise ratio, with virtually no leaky expression.
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Through incorporation into
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<BR><BR><BR>
<BR><BR><BR>
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[[Image:arfp_spun_down.jpg|300px|left]][[Image:arfp_spun_down_fluorescence.jpg|300px|right]]
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[[Image:arfp.jpg|center]]
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<center>[wtRFP in SupF-] [ARFP in SupF-] [wtRFP in SupF+] [ARFP in SupF+]</center>
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 +
The Amber suppressed Red Florescent Protein (ARFP) has no virtually expression in SupF- (2) cells while visually apparent levels of red pigment is present in SupF+ cells (4). ARFP expression was compared against wtRFP expression in both cell types (1 and 3).
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[[Team:Rice_University/OUR TEAM|OUR TEAM]]  ::: 
 +
[[Team:Rice_University|SUMMARY]] :::  [[Team:Rice_University/BACKGROUND|INTRODUCTION]] :::  [[Team:Rice_University/STRATEGY|STRATEGY]] :::  [[Team:Rice_University/RESULTS|RESULTS]] :::  [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]] :::  [[Team:Rice_University/GALLERY|GALLERY]]
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Latest revision as of 19:41, 16 January 2009


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OUR TEAM:::SUMMARY ::: BACKGROUND ::: STRATEGY ::: CONSTRUCTS ::: RESULTS ::: ONGOING WORK

Constructs

Saccharomyces cerevisiae is widely used for baking and brewing, a versatile eukaryotic model system, and particularly useful for synthesizing metabolites under fermentation conditions. The microaerobic conditions of fermentation impede the oxidation of sensitive bioreactive compounds and are optimal for the de novo synthesis of resveratrol. To achieve our project goals and expand the yeast synthetic biology toolbox, we have constructed BioBricks encoding 3 yeast promoters, 3 yeast terminators, a 2-micron origin of replication, 2 selectable markers, 2 metabolic enzymes, and a yeast integration plasmid. We have also submitted two additional parts representing foundational tools, including a gene encoding an amber suppressed RFP biobrick for screening of SupF+ (Amber suppressor) genotype and an amber suppressor tRNA biobrick.


Yeast Promoters

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 BBa_K122000] pPGK1 ~1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation. 1497bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122002 BBa_K122002] pADH1 700bp upstream of ADH1 promoter region. Constitutive promoter under aerobic and anaerobic conditions. 701bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122017] pGAL1 + tetO Glucose repressible / galactose inducible GAL1 promoter. An additional TetR operator site was included to allow repression by TetR. 484bp


Yeast Terminators

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 BBa_K122003] tCYC1 300bp downstream the CYC1 coding region in a standard yeast strain. 300bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 BBa_K122004] tADH1 300bp downstream the ADH1 coding region in a standard yeast strain. 300bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122013 BBa_K122013] tPGK1 1000bp downstream the PGK1 coding region in an industrial yeast strain. 1000bp

 


Selectable Markers

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122018] ZeoR Zeocin/Bleocin Resistance Gene 300bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122008 BBa_K122008] BleoR Bleocin Resistance Gene under pTet promoter 80bp-ish
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122014 BBa_K122014] ORI+HisTag 2 Micron ORI and auxotrophic histidine marker <9000bp

 



Project Specific Constructs

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122001] [pGAL1][tetO][ZeoR] C2.jpg 874bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005] Tyrosine Ammonia Lyase TAL.jpg 1933bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010] 4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein 4CL.jpg 4000bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 BBa_K122012] [pPGK1][4CL:STS][tCYC1] C1.jpg 5497bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 BBa_K122015] [pGAL1][tetO][ZeoR][tADH1] C2full.jpg 1175bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 BBa_K122021] [pADH1][TAL][tPGK1] C3.jpg 2651bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122019 BBa_K122019] [pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1] C1C2C3.jpg 1824bp

 


Additional Bacterial Parts


Novel Zero Leak Inverter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006])

Amber Suppressor tRNA
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007] The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons. Charges with tyrosine. 211bp
[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006] Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation. 923bp



Through incorporation of amber stop codons or point mutations of tyrosine codons (TAC) to TAG within the coding region, a genetic circuit can be used to add an additional level of regulation and determine whether a full protein or partial peptide with be synthesized. This design has high signal-to-noise ratio, with virtually no leaky expression.




Arfp.jpg

The Amber suppressed Red Florescent Protein (ARFP) has no virtually expression in SupF- (2) cells while visually apparent levels of red pigment is present in SupF+ cells (4). ARFP expression was compared against wtRFP expression in both cell types (1 and 3).



OUR TEAM  ::: SUMMARY ::: INTRODUCTION ::: STRATEGY ::: RESULTS ::: ONGOING WORK ::: GALLERY

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