Team:Rice University/CONSTRUCTS

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Latest revision as of 19:41, 16 January 2009

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

BBa_K122000	pPGK1	~1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation.	1497bp
BBa_K122002	pADH1	700bp upstream of ADH1 promoter region. Constitutive promoter under aerobic and anaerobic conditions.	701bp
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

BBa_K122003	tCYC1	300bp downstream the CYC1 coding region in a standard yeast strain.	300bp
BBa_K122004	tADH1	300bp downstream the ADH1 coding region in a standard yeast strain.	300bp
BBa_K122013	tPGK1	1000bp downstream the PGK1 coding region in an industrial yeast strain.	1000bp

Selectable Markers

BBa_K122018	ZeoR	Zeocin/Bleocin Resistance Gene	300bp
BBa_K122008	BleoR	Bleocin Resistance Gene under pTet promoter	80bp-ish
BBa_K122014	ORI+HisTag	2 Micron ORI and auxotrophic histidine marker	<9000bp

Project Specific Constructs

BBa_K122001	[pGAL1][tetO][ZeoR]	874bp
BBa_K122005	Tyrosine Ammonia Lyase	1933bp
BBa_K122010	4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein	4000bp
BBa_K122012	[pPGK1][4CL:STS][tCYC1]	5497bp
BBa_K122015	[pGAL1][tetO][ZeoR][tADH1]	1175bp
BBa_K122021	[pADH1][TAL][tPGK1]	2651bp
BBa_K122019	[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1]	1824bp

Additional Bacterial Parts

Novel Zero Leak Inverter (K122007 and K122006)

Amber Suppressor tRNA

K122007	The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons. Charges with tyrosine.	211bp
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.

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

@@ Line 11: / Line 11: @@
-[[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]] :::
+[[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]]
 {| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}
@@ Line 17: / Line 18: @@
 |
- <h1> Constructs</h1>
+==='''Constructs'''===
-     <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 />
+     <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>
-    <h2>Yeast Regulator Biobricks</h2>
-    <p>Three unique constitutive and repressible yeast promoters were introduced into the registry. </p>
-    <table width="756" border="1">
+<table width="756" border="1">
-   <caption>
+   <h2>
          Yeast Promoters
-       </caption>
+       </h2>
        <tr>
          <td width="120">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 BBa_K122000]</td>
          <td width="118">pPGK1</td>
-         <td width="466">This is the 1500 bp upstream of the PGK1 coding region in an industrial yeast strain. Constitutive promoter. </td>
+         <td width="466">~1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation. </td>
-         <td width="24">1497</td>
+         <td width="24">1497bp</td>
        </tr>
        <tr>
          <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122002 BBa_K122002]</td>
          <td>pADH1</td>
-         <td>700bp upstream of ADH1 promoter region  containing RBS. Constitutive promoter.</td>
+         <td>700bp upstream of ADH1 promoter region. Constitutive promoter under aerobic and anaerobic conditions.</td>
-         <td>701</td>
+         <td>701bp</td>
        </tr>
        <tr>
          <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122017]</td>
          <td>pGAL1 + tetO</td>
-         <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>
+         <td>Glucose repressible / galactose inducible GAL1 promoter.  An additional TetR operator site was included to allow repression by TetR.</td>
-         <td>484</td>
+         <td>484bp</td>
        </tr>
-     </table><BR>
+     </table>
-    <p>Four additional yeast terminators were introduced into the registry. </p>
      <table width="756" border="1">
-      <caption>
+      <h2>
          Yeast Terminators
-       </caption> <tr>
+       </h2> <tr>
          <td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 BBa_K122003]</td>
          <td width="74">tCYC1</td>
          <td width="493">300bp downstream the CYC1 coding region in a standard yeast strain.</td>
-         <td width="46">300</td>
+         <td width="46">300bp</td>
        </tr>
        <tr>
@@ Line 59: / Line 61: @@
          <td>tADH1</td>
          <td>300bp downstream the ADH1 coding region in a standard yeast strain.</td>
-         <td>300</td>
+         <td>300bp</td>
        </tr>
        <tr>
@@ Line 65: / Line 67: @@
          <td>tPGK1</td>
          <td>1000bp downstream the PGK1 coding region in an industrial yeast strain.</td>
-         <td>1000</td>
+         <td>1000bp</td>
        </tr>
      </table>
      <p>&nbsp;</p><BR>
-    <h2>Selectable Markers</h2>
      <table width="756" border="1">
-       <caption>
+       <h2>
          Selectable Markers
-       </caption>
+       </h2>
        <tr>
          <td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122018]</td>
          <td width="74">ZeoR</td>
-         <td width="493">Zeocin Resistance Gene</td>
+         <td width="493">Zeocin/Bleocin Resistance Gene</td>
-         <td width="46">300</td>
+         <td width="46">300bp</td>
        </tr>
        <tr>
@@ Line 84: / Line 85: @@
          <td>BleoR</td>
          <td>Bleocin Resistance Gene under pTet promoter</td>
-         <td>800ish</td>
+         <td>80bp-ish</td>
        </tr>
        <tr>
          <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122014 BBa_K122014]</td>
          <td>ORI+HisTag</td>
-         <td>2 Micron ORI and Histadine Tag </td>
+         <td>2 Micron ORI and auxotrophic histidine marker </td>
-         <td>&lt;9000</td>
+         <td>&lt;9000bp</td>
        </tr>
      </table>
@@ Line 96: / Line 97: @@
      <h2>Project Specific Constructs</h2>
-This year's project involved the integration of 4-coumarate:coenzyme A ligase/ stilbene synthase fusion protein, a tyrosine ammonia lyase, and a zeocin resistance selectable marker. For more information, please visit [[Team:Rice_University/STRATEGY|Strategy]].
-<BR>
      <table width="756" border="1">
        <tr>
@@ Line 103: / Line 102: @@
          <td width="451">[pGAL1][tetO][ZeoR]</td>
          <td width="74">[[Image:C2.jpg|120px]]</td>
-         <td width="90">&nbsp;</td>
+         <td width="90">874bp</td>
        </tr>
        <tr>
@@ Line 109: / Line 108: @@
          <td>Tyrosine Ammonia Lyase</td>
          <td>[[Image:TAL.jpg|50px]]</td>
-         <td>&nbsp;</td>
+         <td>1933bp</td>
        </tr>
        <tr>
          <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]</td>
-         <td>4CL:STS</td>
+         <td>4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein</td>
          <td>[[Image:4CL.jpg|50px]]</td>
-         <td>&nbsp;</td>
+         <td>4000bp</td>
        </tr>
        <tr>
          <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 BBa_K122012]</td>
          <td>[pPGK1][4CL:STS][tCYC1]</td>
-         <td>&nbsp;</td>
+         <td>[[Image:C1.jpg|150px]]</td>
-         <td>&nbsp;</td>
+         <td>5497bp</td>
        </tr>
        <tr>
          <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 BBa_K122015]</td>
          <td>[pGAL1][tetO][ZeoR][tADH1]</td>
-         <td>&nbsp;</td>
+         <td>[[Image:C2full.jpg|150px]]</td>
-         <td>&nbsp;</td>
+         <td>1175bp</td>
        </tr>
        <tr>
-         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122016 BBa_K122016]</td>
+         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 BBa_K122021]</td>
-         <td>4CL:STS with CYC1 terminator.</td>
+         <td>[pADH1][TAL][tPGK1]</td>
-         <td>&nbsp;</td>
+         <td>[[Image:C3.jpg|150px]]</td>
-         <td>&nbsp;</td>
+         <td>2651bp</td>
        </tr>
        <tr>
-         <td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122016 BBa_K122019]</td>
+         <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>[[Image:C1C2C3.jpg|350px]]</td>
-         <td>&nbsp;</td>
+         <td>1824bp</td>
        </tr>
      </table>
@@ Line 145: / Line 144: @@
      <h2>Additional Bacterial Parts</h2>
 <BR>
-Novel Zero Leak Inverter Concept
+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])
-[[Image:arfp_spun_down.jpg|300px|left]]
-[[Image:arfp_spun_down_fluorescence.jpg|300px|center]]
-<center>wtRFP in SupF- / ARFP in SupF- /wtRFP in SupF+ / ARFP in SupF+</center>
+[[Image:tRNA.png|thumb|250px|left|Amber Suppressor tRNA]]
+<table width="477" border="1">
+      <tr>
+        <td width="71">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007]</td>
+        <td width="354">The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons.  Charges with tyrosine.</td>
+        <td width="30">211bp</td>
+      </tr>
+      <tr>
+        <td height="54">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006]</td>
+        <td>Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation.</td>
+        <td>923bp</td>
+      </tr>
+    </table>
-<BR>
+<BR><BR>
-    <h2><br />
-    </h2><BR>
+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.
-|}
+<BR><BR><BR>
+[[Image:arfp.jpg|center]]
+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).
+|}
+[[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]]
 |}