http://2008.igem.org/wiki/index.php?title=Special:Contributions/Pnguyen&feed=atom&limit=50&target=Pnguyen&year=&month=2008.igem.org - User contributions [en]2024-03-29T06:29:29ZFrom 2008.igem.orgMediaWiki 1.16.5http://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T05:22:20Z<p>Pnguyen: /* C. HPLC Data */</p>
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<div><!--MAIN PROJECT PAGE--><br />
{|<br />
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[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
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[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol without the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|700px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|700px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|700px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|700px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of ''p''-coumaric acid and 20ug/mL of ''trans''-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|700px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|700px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve.''' Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<BR><br />
<BR><br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C7. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T05:19:25Z<p>Pnguyen: /* C. HPLC Data */</p>
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<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol without the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of ''p''-coumaric acid and 20ug/mL of ''trans''-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve.''' Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<BR><br />
<BR><br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C7. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/CONSTRUCTSTeam:Rice University/CONSTRUCTS2008-10-30T05:16:57Z<p>Pnguyen: /* Constructs */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]]<BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-indent: 15pt;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
<br />
==='''Constructs'''===<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 /><br />
</p><br />
<br />
<br />
<table width="756" border="1"><br />
<h2><br />
Yeast Promoters<br />
</h2><br />
<tr><br />
<td width="120">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 BBa_K122000]</td><br />
<td width="118">pPGK1</td><br />
<td width="466">~1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation. </td><br />
<td width="24">1497bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122002 BBa_K122002]</td><br />
<td>pADH1</td><br />
<td>700bp upstream of ADH1 promoter region. Constitutive promoter under aerobic and anaerobic conditions.</td><br />
<td>701bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122017]</td><br />
<td>pGAL1 + tetO</td><br />
<td>Glucose repressible / galactose inducible GAL1 promoter. An additional TetR operator site was included to allow repression by TetR.</td><br />
<td>484bp</td><br />
</tr><br />
</table><br />
<br />
<br />
<table width="756" border="1"><br />
<h2><br />
Yeast Terminators<br />
</h2> <tr><br />
<td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 BBa_K122003]</td><br />
<td width="74">tCYC1</td><br />
<td width="493">300bp downstream the CYC1 coding region in a standard yeast strain.</td><br />
<td width="46">300bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 BBa_K122004]</td><br />
<td>tADH1</td><br />
<td>300bp downstream the ADH1 coding region in a standard yeast strain.</td><br />
<td>300bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122013 BBa_K122013]</td><br />
<td>tPGK1</td><br />
<td>1000bp downstream the PGK1 coding region in an industrial yeast strain.</td><br />
<td>1000bp</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><BR><br />
<table width="756" border="1"><br />
<h2><br />
Selectable Markers<br />
</h2><br />
<tr><br />
<td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122018]</td><br />
<td width="74">ZeoR</td><br />
<td width="493">Zeocin/Bleocin Resistance Gene</td><br />
<td width="46">300bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122008 BBa_K122008]</td><br />
<td>BleoR</td><br />
<td>Bleocin Resistance Gene under pTet promoter</td><br />
<td>80bp-ish</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122014 BBa_K122014]</td><br />
<td>ORI+HisTag</td><br />
<td>2 Micron ORI and auxotrophic histidine marker </td><br />
<td>&lt;9000bp</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><BR><BR><br />
<h2>Project Specific Constructs</h2><br />
<br />
<table width="756" border="1"><br />
<tr><br />
<td width="113">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122001]</td><br />
<td width="451">[pGAL1][tetO][ZeoR]</td><br />
<td width="74">[[Image:C2.jpg|120px]]</td><br />
<td width="90">874bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005]</td><br />
<td>Tyrosine Ammonia Lyase</td><br />
<td>[[Image:TAL.jpg|50px]]</td><br />
<td>1933bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]</td><br />
<td>4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein</td><br />
<td>[[Image:4CL.jpg|50px]]</td><br />
<td>4000bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 BBa_K122012]</td><br />
<td>[pPGK1][4CL:STS][tCYC1]</td><br />
<td>[[Image:C1.jpg|150px]]</td><br />
<td>5497bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 BBa_K122015]</td><br />
<td>[pGAL1][tetO][ZeoR][tADH1]</td><br />
<td>[[Image:C2full.jpg|150px]]</td><br />
<td>1175bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 BBa_K122021]</td><br />
<td>[pADH1][TAL][tPGK1]</td><br />
<td>[[Image:C3.jpg|150px]]</td><br />
<td>2651bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122016 BBa_K122019]</td><br />
<td>[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1]</td><br />
<td>[[Image:C1C2C3.jpg|350px]]</td><br />
<td>1824bp</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><BR><br />
<h2>Additional Bacterial Parts</h2><br />
<BR><br />
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])<br />
<br />
[[Image:tRNA.png|thumb|250px|left|Amber Suppressor tRNA]]<br />
<table width="477" border="1"><br />
<tr><br />
<td width="71">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007]</td><br />
<td width="354">The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons. Charges with tyrosine.</td><br />
<td width="30">211bp</td><br />
</tr><br />
<tr><br />
<td height="54">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006]</td><br />
<td>Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation.</td><br />
<td>923bp</td><br />
</tr><br />
</table><br />
<br />
<BR><BR><br />
<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><BR><BR><br />
[[Image:arfp.jpg|center]]<br />
<br />
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). <br />
<br />
<br />
<br />
<br />
<br />
|}<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]]<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/CONSTRUCTSTeam:Rice University/CONSTRUCTS2008-10-30T05:15:48Z<p>Pnguyen: /* Constructs */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
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[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
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{|<br />
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[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
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<br />
==='''Constructs'''===<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 /><br />
</p><br />
<br />
<br />
<table width="756" border="1"><br />
<h2><br />
Yeast Promoters<br />
</h2><br />
<tr><br />
<td width="120">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 BBa_K122000]</td><br />
<td width="118">pPGK1</td><br />
<td width="466">~1500 bp upstream of the PGK1 coding region. Strongly induced during fermentation. </td><br />
<td width="24">1497bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122002 BBa_K122002]</td><br />
<td>pADH1</td><br />
<td>700bp upstream of ADH1 promoter region. Constitutive promoter under aerobic and anaerobic conditions.</td><br />
<td>701</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122017]</td><br />
<td>pGAL1 + tetO</td><br />
<td>Glucose repressible / galactose inducible GAL1 promoter. An additional TetR operator site was included to allow repression by TetR.</td><br />
<td>484bp</td><br />
</tr><br />
</table><br />
<br />
<br />
<table width="756" border="1"><br />
<h2><br />
Yeast Terminators<br />
</h2> <tr><br />
<td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 BBa_K122003]</td><br />
<td width="74">tCYC1</td><br />
<td width="493">300bp downstream the CYC1 coding region in a standard yeast strain.</td><br />
<td width="46">300bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 BBa_K122004]</td><br />
<td>tADH1</td><br />
<td>300bp downstream the ADH1 coding region in a standard yeast strain.</td><br />
<td>300bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122013 BBa_K122013]</td><br />
<td>tPGK1</td><br />
<td>1000bp downstream the PGK1 coding region in an industrial yeast strain.</td><br />
<td>1000bp</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><BR><br />
<table width="756" border="1"><br />
<h2><br />
Selectable Markers<br />
</h2><br />
<tr><br />
<td width="115">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122018]</td><br />
<td width="74">ZeoR</td><br />
<td width="493">Zeocin/Bleocin Resistance Gene</td><br />
<td width="46">300bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122008 BBa_K122008]</td><br />
<td>BleoR</td><br />
<td>Bleocin Resistance Gene under pTet promoter</td><br />
<td>80-bp ish</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122014 BBa_K122014]</td><br />
<td>ORI+HisTag</td><br />
<td>2 Micron ORI and auxotrophic histidine marker </td><br />
<td>&lt;9000bp</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><BR><BR><br />
<h2>Project Specific Constructs</h2><br />
<br />
<table width="756" border="1"><br />
<tr><br />
<td width="113">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 BBa_K122001]</td><br />
<td width="451">[pGAL1][tetO][ZeoR]</td><br />
<td width="74">[[Image:C2.jpg|120px]]</td><br />
<td width="90">874</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005]</td><br />
<td>Tyrosine Ammonia Lyase</td><br />
<td>[[Image:TAL.jpg|50px]]</td><br />
<td>1933bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]</td><br />
<td>4-coumarate CoA ligase :: Stilbene Synthase Fusion Protein</td><br />
<td>[[Image:4CL.jpg|50px]]</td><br />
<td>4000bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 BBa_K122012]</td><br />
<td>[pPGK1][4CL:STS][tCYC1]</td><br />
<td>[[Image:C1.jpg|150px]]</td><br />
<td>5497bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 BBa_K122015]</td><br />
<td>[pGAL1][tetO][ZeoR][tADH1]</td><br />
<td>[[Image:C2full.jpg|150px]]</td><br />
<td>1175bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 BBa_K122021]</td><br />
<td>[pADH1][TAL][tPGK1]</td><br />
<td>[[Image:C3.jpg|150px]]</td><br />
<td>2651bp</td><br />
</tr><br />
<tr><br />
<td>[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122016 BBa_K122019]</td><br />
<td>[pPGK1][4CL:STS][tCYC1][pGAL1][tetO][ZeoR][tADH1]</td><br />
<td>[[Image:C1C2C3.jpg|350px]]</td><br />
<td>1824bp</td><br />
</tr><br />
</table><br />
<p>&nbsp;</p><BR><br />
<h2>Additional Bacterial Parts</h2><br />
<BR><br />
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])<br />
<br />
[[Image:tRNA.png|thumb|250px|left|Amber Suppressor tRNA]]<br />
<table width="477" border="1"><br />
<tr><br />
<td width="71">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122007 K122007]</td><br />
<td width="354">The supF construct, an amber suppressor tRNA, allows for read-through at native amber (TAG) stop codons. Charges with tyrosine.</td><br />
<td width="30">211</td><br />
</tr><br />
<tr><br />
<td height="54">[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122006 K122006]</td><br />
<td>Point Mutation of RFP(13521) with incorporation of an amber stop codon at the native tyrosine required for fluorophore maturation.</td><br />
<td>923</td><br />
</tr><br />
</table><br />
<br />
<BR><BR><br />
<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><BR><BR><br />
[[Image:arfp.jpg|center]]<br />
<br />
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). <br />
<br />
<br />
<br />
<br />
<br />
|}<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]]<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/STRATEGYTeam:Rice University/STRATEGY2008-10-30T05:09:27Z<p>Pnguyen: </p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]]<BR><br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: [[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
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{|align="justify" style="background-color:#FFFFFF;15pt;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
===Pathway Design for Resveratrol Biosynthesis===<br />
[[Image:TAL.png|right|190px|thumb|[http://www.rcsb.org/pdb/explore.do?structureId=1T6P TAL] monomer.]]<br />
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=169745 Tyrosine Ammonia-Lyase] (TAL, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]) - TAL catalyzes the conversion of L-tyrosine to ''p''-coumaric acid and ammonia. TAL also exhibits Phenylalanine Ammonia-Lyase (PAL) activity, converting L-phenylalanine to ''trans''-cinnamic acid and ammonia. Our work has focused on using [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=36575&Template=fungiYeast ''Rhodotorula glutinis''] TAL because its ratio of TAL to PAL activity is high compared to other TAL homologs. In addition, previous studies have shown that this enzyme can be expressed as a functional protein in ''Saccharomyces cerevisiae'' and ''Escherichia coli''. While the ''p''-coumaric acid produced by TAL will serve as a substrate for resveratrol biosynthesis, the ''trans''-cinnamic acid is expected to add a "floral" and "honey-like" bouquet to the beer. <br />
[[Image:TAL catalysis.png|left|500px]]<br />
<br />
<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><br />
<br />
[[Image:STS.png|right|190px|thumb|[http://www.rcsb.org/pdb/explore.do?structureId=1Z1F Peanut STS] monomer bound to resveratrol.]]<br />
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NM_001084228.1 4-coumarate CoA ligase] :: [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?tool=portal&db=nuccore&term=&query_key=18&dopt=gb&dispmax=20&page=1&qty=1&WebEnv=1BAyWdNufvUWxpNM-oKD-9JoRgEPTXzU_kF02A2hfcePWB3nxyPvHO3gqlDJksRdZq9GmTDHNDmKEFuabzP4VKB%40263F5D1B8F754EA0_0062SID&WebEnvRq=1 Stilbene Synthase] Fusion Protein (4CL:STS, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005]) - This enzyme fusion is comprised of ''Arabidopsis thaliana'' 4-coumarate-CoA ligase (4CL), which catalyzes the conversion of ''p''-coumaric acid to 4-coumaroyl-CoA, and ''Vitis vinifera'' Stilbene Synthase, which catalyzes the condensation of resveratrol from 4-coumaroyl-CoA and three malonyl-CoA molecules. This 4CL:STS fusion protein was selected for our project because it has been shown to more efficiently produce resveratrol than coexpression of the proteins separately (possibly due to substrate channeling), see Zhang Y, et al. (2006) Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and Mammalian cells. ''J Am Chem Soc.'' '''128'''(40):13030-1.<br />
.<br />
[[Image:4CL_STS catalysis.png|left|550px]]<br />
<br />
<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><br />
<br />
=== Circuit Design for Recombination in Yeast===<br />
[[Image:circuit.png|center|700px|thumb|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122020 BBa_K122020].]]<br />
*Our design goal was to construct a circuit that would propagate through several generations without a selection pressure and be highly expressed during all stages of fermentation. To address these goals, we constructed three expression cassettes that, when concatenated, would integrate genomically into a highly transcribed locus, have an inducible selectable marker, and highly express the resveratrol pathway under anaerobic conditions.<br />
<BR><br />
<br />
{|align="left" style="border-color:#FF0000;text-align:left" border="10" cellpadding="0" width="29%"<br />
|<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 Cassette 1] - Anaerobically induced expression of 4CL:STS<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 pPGK1] - Serves the dual purpose of 5' homologous recombination region and anaerobically induced promoter.<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 4CL:STS] - Converts ''p''-coumaric acid and 3 malonyl-CoA molecules to resveratrol. <br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 tCYC1] - Bi-directional transcriptional stop.<br />
|}<br />
{|align="left" style="border-color:#33CC33;text-align:left" border="10" cellpadding="0" width="34%"<br />
|<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 Cassette 2] - Inducible expression of Bleocin resistance.<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 pGAL1 TetO ZeoR] - Provides galactose inducible, TetR repressible, Zeocin/Bleocin resistance. This part will allow us to select for recombinants. <br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 tADH1] - Bi-directional transcriptional stop. Strategically placed downstream of ZeoR and upstream of pADH1 to prevent erroneous recombination at those regions. <br />
|}<br />
{|align="left" style="border-color:#3333CC;text-align:left" border="10" cellpadding="0" width="33%"<br />
|<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 Cassette 3] - Constitutive expression of TAL<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 pADH1] - Constitutive promoter. Strategically placed to prevent erroneous recombination with tADH1. <br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 TAL] - Converts L-tyrosine to ''p''-coumaric acid and L-phenylalanine to ''trans''-cinnamic acid.<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122013 tPGK1] - Dual purpose of 3' homologous recombination region and bi-directional transcriptional terminator.<br />
|}<br />
<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><br />
<br />
===Selection of Brewing Strain ===<br />
[[Image: St_Arnold.png|left|200px|thumb|[http://www.saintarnold.com/ http://www.saintarnold.com/]]]<br />
{|align="left" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="45%"<br />
|<br />
*Our cellular brewing chassis can be best described by its use in St. Arnold's hefeweizen beer. This is top-fermenting yeast which causes a great deal of gas induced convection during primary brewing. This will help to keep our yeast suspended and well-mixed. Additionally, hefeweizen beers are commonly served unfiltered, allowing for the consumption of whole yeast cells. We postulate this will dramatically increase the amount of resveratrol available for consumption. Hefeweizen beers are said to have "light citrus notes over a full bodied breadiness". <br />
|}<br />
[[Image: SAB_Hefe.png|right|175px]]<br />
<br />
<br />
<br />
<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><br />
<br />
<br />
|}<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/STRATEGYTeam:Rice University/STRATEGY2008-10-30T05:07:46Z<p>Pnguyen: </p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]]<BR><br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: [[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
{|align="justify" style="background-color:#FFFFFF;15pt;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
===Pathway Design for Resveratrol Biosynthesis===<br />
[[Image:TAL.png|right|190px|thumb|[http://www.rcsb.org/pdb/explore.do?structureId=1T6P TAL] monomer.]]<br />
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=169745 Tyrosine Ammonia-Lyase] (TAL, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122010 BBa_K122010]) - TAL catalyzes the conversion of L-tyrosine to ''p''-coumaric acid and ammonia. TAL also exhibits Phenylalanine Ammonia-Lyase (PAL) activity, converting L-phenylalanine to ''trans''-cinnamic acid and ammonia. Our work has focused on using [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=36575&Template=fungiYeast ''Rhodotorula glutinis''] TAL because its ratio of TAL to PAL activity is high compared to other TAL homologs. In addition, previous studies have shown that this enzyme can be expressed as a functional protein in ''Saccharomyces cerevisiae'' and ''Escherichia coli''. While the ''p''-coumaric acid produced by TAL will serve as a substrate for resveratrol biosynthesis, the ''trans''-cinnamic acid is expected to add a "floral" and "honey-like" bouquet to the beer. <br />
[[Image:TAL catalysis.png|left|500px]]<br />
<br />
<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><br />
<br />
[[Image:STS.png|right|190px|thumb|[http://www.rcsb.org/pdb/explore.do?structureId=1Z1F Peanut STS] monomer bound to resveratrol.]]<br />
*[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NM_001084228.1 4-coumarate CoA ligase] :: [http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?tool=portal&db=nuccore&term=&query_key=18&dopt=gb&dispmax=20&page=1&qty=1&WebEnv=1BAyWdNufvUWxpNM-oKD-9JoRgEPTXzU_kF02A2hfcePWB3nxyPvHO3gqlDJksRdZq9GmTDHNDmKEFuabzP4VKB%40263F5D1B8F754EA0_0062SID&WebEnvRq=1 Stilbene Synthase] Fusion Protein (4CL:STS, [http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 BBa_K122005]) - This enzyme fusion is comprised of ''Arabidopsis thaliana'' 4-coumarate-CoA ligase (4CL), which catalyzes the conversion of ''p''-coumaric acid to 4-coumaroyl-CoA, and ''Vitis vinifera'' Stilbene Synthase, which catalyzes the condensation of resveratrol from 4-coumaroyl-CoA and three malonyl-CoA molecules. This 4CL:STS fusion protein was selected for our project because it has been shown to more efficiently produce resveratrol than coexpression of the proteins separately (possibly due to substrate channeling), see Zhang Y, et al. (2006) Using unnatural protein fusions to engineer resveratrol biosynthesis in yeast and<br />
Mammalian cells. ''J Am Chem Soc.'' '''128'''(40):13030-1.<br />
PMID: 17017764 [PubMed - indexed for MEDLINE]<br />
.<br />
[[Image:4CL_STS catalysis.png|left|550px]]<br />
<br />
<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><br />
<br />
=== Circuit Design for Recombination in Yeast===<br />
[[Image:circuit.png|center|700px|thumb|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122020 BBa_K122020].]]<br />
*Our design goal was to construct a circuit that would propagate through several generations without a selection pressure and be highly expressed during all stages of fermentation. To address these goals, we constructed three expression cassettes that, when concatenated, would integrate genomically into a highly transcribed locus, have an inducible selectable marker, and highly express the resveratrol pathway under anaerobic conditions.<br />
<BR><br />
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|<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122012 Cassette 1] - Anaerobically induced expression of 4CL:STS<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122000 pPGK1] - Serves the dual purpose of 5' homologous recombination region and anaerobically induced promoter.<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 4CL:STS] - Converts ''p''-coumaric acid and 3 malonyl-CoA molecules to resveratrol. <br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122003 tCYC1] - Bi-directional transcriptional stop.<br />
|}<br />
{|align="left" style="border-color:#33CC33;text-align:left" border="10" cellpadding="0" width="34%"<br />
|<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122015 Cassette 2] - Inducible expression of Bleocin resistance.<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122001 pGAL1 TetO ZeoR] - Provides galactose inducible, TetR repressible, Zeocin/Bleocin resistance. This part will allow us to select for recombinants. <br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 tADH1] - Bi-directional transcriptional stop. Strategically placed downstream of ZeoR and upstream of pADH1 to prevent erroneous recombination at those regions. <br />
|}<br />
{|align="left" style="border-color:#3333CC;text-align:left" border="10" cellpadding="0" width="33%"<br />
|<br />
*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122021 Cassette 3] - Constitutive expression of TAL<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122004 pADH1] - Constitutive promoter. Strategically placed to prevent erroneous recombination with tADH1. <br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122005 TAL] - Converts L-tyrosine to ''p''-coumaric acid and L-phenylalanine to ''trans''-cinnamic acid.<br />
*#[http://partsregistry.org/wiki/index.php?title=Part:BBa_K122013 tPGK1] - Dual purpose of 3' homologous recombination region and bi-directional transcriptional terminator.<br />
|}<br />
<BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><BR><br />
<br />
===Selection of Brewing Strain ===<br />
[[Image: St_Arnold.png|left|200px|thumb|[http://www.saintarnold.com/ http://www.saintarnold.com/]]]<br />
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*Our cellular brewing chassis can be best described by its use in St. Arnold's hefeweizen beer. This is top-fermenting yeast which causes a great deal of gas induced convection during primary brewing. This will help to keep our yeast suspended and well-mixed. Additionally, hefeweizen beers are commonly served unfiltered, allowing for the consumption of whole yeast cells. We postulate this will dramatically increase the amount of resveratrol available for consumption. Hefeweizen beers are said to have "light citrus notes over a full bodied breadiness". <br />
|}<br />
[[Image: SAB_Hefe.png|right|175px]]<br />
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[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/BACKGROUNDTeam:Rice University/BACKGROUND2008-10-30T05:00:04Z<p>Pnguyen: /* History of Resveratrol */</p>
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[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: [[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
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<!--BACKGROUND CONTENT BELOW--><br />
<br />
=== '''History of Resveratrol''' ===<br />
[[Image:trans-resveratrol.jpg|left|frame|none|trans-Resveratrol]]<br />
In 1940, resveratrol was identified as the active component in ''Cassia quniquangulata'' (cinnamon) extract and was shown to be responsible for the anti-inflammatory properties of ''Polygonum cuspidatum'' root, which is used in traditional Chinese and Japanese medicine [1]. Since then, studies have shown that resveratrol is a member of a class of compounds called phytoalexins, which plants use as a defense mechanism against pathogens [2]. Grapes and several types of berries, including blueberries, bilberries, and cranberries, produce appreciable levels of resveratrol, and thus the main sources of resveratrol in the human diet are wine and juices [3]. The discovery of resveratrol in wine [4] implicated a role for this compound in the “French Paradox," the observation that the French exhibit a relatively low rate of cardiovascular disease although their diet is high in saturated fats.<br />
<br />
==='''Health Benefits of Resveratrol''' ===<br />
[[Image:RSV.jpg|right|thumb|300px|History of Resveratrol Citatitions on PubMed ]]<br />
A PubMed search for “resveratrol” indicates that scientific interest in the health benefits of this phytoalexin is growing exponentially. To date, a diverse array of health benefits have been associated with resveratrol, including:<br />
<br />
<br />
<B>1. Improved insulin sensitivity.</B> Resveratrol has been shown as a potent therapeutic for type 2 diabetes [5]. Pharmaceuticals based on resveratrol-like compounds for the treatment of diabetes are currently in Phase I clinical trials [6].<br />
<br />
<B>2. Inhibition of carcinogenesis.</B> Resveratrol induces cell death specifically in cancerous cells. This property has been demonstrated for a variety of cancers, including colon [7-9], pancreatic [10], prostate [11,12], breast [13,14], and skin [15,16] cancers. Several ongoing Phase I human clinical trials are investigating the use of resveratrol as a cancer therapy [17,18]<br />
<br />
<B>3. Extended lifespan.</B> Resveratrol mimics the effects of caloric restriction in mammals and has been shown to extend lifespans in invertebrates [19,20], and a fish model [21]. In addition, resveratrol reduces the genetic changes associated with aging in a mammalian mouse model [22-24]. PGC-1alpha levels were decreased threefold by induction of genes for oxidative phosphorylation and mitochondrial biogenesis in wt SIRT1 mice but no change in SIRT1(-/-) MEFs [25].<br />
<br />
<B>4. Improved cardiovascular function.</B> Resveratrol exhibits cardioprotective effects, such as suppression of atherosclerosis, inhibition of platelet aggregation, promotion of vasorelaxation, and modulation of triglyceride blood levels [22,26-28].<br />
<br />
<B>5. Reduced Neurodegeneration.</B> Mouse models of Alzheimer's and Parkinson's disease have provided evidence that resveratrol functions as a protective agent against degenerative neural diseases [29,30]. In addition, experiments with rats, mice, and gerbils show that resveratrol administration protects against brain damage following ischemic stroke [31-33].<br />
<br />
The biochemical origin of the diverse pharmacological effects of resveratrol is not completely understood, but a variety of mechanisms have been proposed for the molecule's action [3]. <br />
<BR><br />
<br />
=== '''Rationale for Creating Resveratrol-Enriched Beer''' ===<br />
<br />
<br />
[[Image:brewing.jpg|left]]<br />
<br />
One of the best sources of resveratrol is wine, with red wine having concentrations ranging from 0.1 to 14.3 mg/L and white wine having concentrations ranging from <0.1 to 2.1 mg/L [3]. Beer is also predicted to contain resveratrol, albeit at lower levels than wine, since hops contains low levels of resveratrol (0.5 to 1 µg/g) [3]. Because beer is more popular in the United States than wine [34], many people consuming alcoholic beverages do not gain the full health benefits of dietary resveratrol. To increase the level of resveratrol present in beer and provide these health benefits for a wider populace, we are working to engineer a brewer’s yeast that synthesizes resveratrol from tyrosine. Like wine, beer is produced under fermentative conditions, and we anticipate that production of resveratrol in beer using our engineered yeast will be an effective method for avoiding air oxidation and inactivation of resveratrol. In addition, we propose that brewer's yeast represents an excellent approach for synthesizing air sensitive prophylactics in beer for widespread and affordable delivery to consumers.<br />
<br />
<BR><BR><br />
<br />
==='''References''' ===<br />
<h6><br />
1: Takaoka, M. J. Of the phenolic substances of white hellebore (Veratrum grandiflorum Loes. Fil.) J. Faculty Sci. Hokkaido Imperial University. 1940; 3: 1-16.<br />
<BR><br />
2. Hammerschmidt, R. PHYTOALEXINS:What Have We Learned After 60 years. Annu. Rev. Phytopathol. 1999; 37:285–306<br />
<BR><br />
3. Baur, J. A. and Sinclair, D. A. Therapeutic potential of resveratrol: the in vivo evidence. Nature Reviews Drug Discovery. 2006; 5: 493-506.<br />
<BR><br />
4. Renaud, S. and Gueguen, R. The French Paradox and wine drinking. Novartis Found. Sympos. 1998; 216:208-217.<br />
<BR><br />
5. Milne JC, et al. Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes. Nature. 2007 Nov 29;450(7170):712-6.<br />
<BR><br />
6. http://www.sirtrispharma.com/pipeline-SRT501.html<br />
<BR><br />
7. Juan ME, et al. Resveratrol induces apoptosis through ROS-dependent mitochondria pathway in HT-29 human colorectal carcinoma cells. J Agric Food Chem. 2008 Jun 25;56(12):4813-8. Epub 2008 Jun 4.<br />
<BR><br />
8. Saiko P, et al. Novel resveratrol analogs induce apoptosis and cause cell cycle arrest in HT29 human colon cancer cells: inhibition of ribonucleotide reductase activity. Oncol Rep. 2008 Jun;19(6):1621-6.<br />
<BR><br />
9. Marel AK, et al. Inhibitory effects of trans-resveratrol analogs molecules on the proliferation and the cell cycle progression of human colon tumoral cells. Mol Nutr Food Res. 2008 May;52(5):538-48.<br />
<BR><br />
10. Bernhaus A, et al. Antitumor effects of KITC, a new resveratrol derivative, in AsPC-1 and BxPC-3 human pancreatic carcinoma cells. Invest New Drugs. 2008 Oct 8. [Epub ahead of print]<br />
<BR><br />
11. Seeni A, et al. Suppression of Prostate Cancer Growth by Resveratrol in The Transgenic Rat for Adenocarcinoma of Prostate (TRAP) Model. Asian Pac J Cancer Prev. 2008 Jan-Mar;9(1):7-14.<br />
<BR><br />
12. Horvath Z, et al. Novel resveratrol derivatives induce apoptosis and cause cell cycle arrest in prostate cancer cell lines. Anticancer Res. 2007 Sep-Oct;27(5A):3459-64.<br />
<BR><br />
13. Filomeni G, et al. trans-Resveratrol induces apoptosis in human breast cancer cells MCF-7 by the activation of MAP kinases pathways. Genes Nutr. 2007 Dec;2(3):295-305. Epub 2007 Oct 18.<br />
<BR><br />
14. Schlachterman A, et al. Combined resveratrol, quercetin, and catechin treatment reduces breast tumor growth in a nude mouse model. Transl Oncol. 2008 Mar;1(1):19-27.<br />
<BR><br />
15. Jang M, Cai L, et al. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science. 1997 Jan 10;275(5297):218-20.<br />
<BR><br />
16. van Ginkel PR, et al. Resveratrol inhibits uveal melanoma tumor growth via early mitochondrial dysfunction. Invest Ophthalmol Vis Sci. 2008 Apr;49(4):1299-306.<br />
<BR><br />
17. http://www.clinicaltrials.gov/ct/show/NCT00256334<br />
<BR><br />
18. http://www.cancer.gov/clinicaltrials/CCUM-2004-0535<br />
<BR><br />
19. Howitz KT, et al. Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature. 2003 Sep 11;425(6954):191-6. Epub 2003 Aug 24.<br />
<BR><br />
20. Wood JG, et al. Sirtuin activators mimic caloric restriction and delay ageing in metazoans. Nature. 2004 Aug 5;430(7000):686-9. <br />
<BR><br />
21. Valenzano DR, et al. Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate. Curr Biol. 2006 Feb 7;16(3):296-300.<br />
<BR><br />
22. Barger JL, et al. A low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice. PLoS ONE. 2008 Jun 4;3(6):e2264.<br />
<BR><br />
23. Kode A, et al. (2007). "Resveratrol induces glutathione synthesis by activation of Nrf2 and protects against cigarette smoke-mediated oxidative stress in human lung epithelial cells". Am J Physiol Lung Cell Mol Physiol. <br />
<BR><br />
24. Baur JA, et al. Resveratrol improves health and survival of mice on a high-calorie diet. Nature. 2006 Nov 16;444(7117):337-42. Epub 2006 Nov 1.<br />
<BR><br />
25. M . Lagouge , C . Argmann , Z . Gerhart-Hines , H . Meziane , C . Lerin , F . Daussin , N . Messadeq , J . Milne , P . Lambert , P . Elliott. Resveratrol Improves Mitochondrial Function and Protects against Metabolic Disease by Activating SIRT1 and PGC-1α . Cell , Volume 127 , Issue 6 , Pages 1109 - 1122 <br />
<BR><br />
26. Samuel SM, et al. Akt/FOXO3a/SIRT1-mediated cardioprotection by n-tyrosol against ischemic stress in rat in vivo model of myocardial infarction: switching gears toward survival and longevity. J Agric Food Chem. 2008 Oct 22;56(20):9692-8. <br />
<BR><br />
27. Hwang JT, et al. Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells. Genes Nutr. 2008 Feb;2(4):323-6.<br />
<BR><br />
28. Gresele P, et al. Resveratrol, at concentrations attainable with moderate wine consumption, stimulates human platelet nitric oxide production.<br />
J Nutr. 2008 Sep;138(9):1602-8.<br />
<BR><br />
29. Kim D, et al. SIRT1 deacetylase protects against neurodegeneration in models for Alzheimer's disease and amyotrophic lateral sclerosis. EMBO J. 2007 Jul 11;26(13):3169-79. Epub 2007 Jun 21.<br />
<BR><br />
30. Jin F, et al. Neuroprotective effect of resveratrol on 6-OHDA-induced Parkinson's disease in rats. Eur J Pharmacol. 2008 Oct 10. [Epub ahead of print]<br />
<BR><br />
31. Sinha K, et al. Protective effect of resveratrol against oxidative stress in middle cerebral artery occlusion model of stroke in rats. Life Sci. 2002 Jun 28;71(6):655-65.<br />
<BR><br />
32. Inoue H, et al. Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice. Neurosci Lett. 2003 Dec 11;352(3):203-6.<br />
<BR><br />
33. Wang Q, et al. Resveratrol protects against global cerebral ischemic injury in gerbils. Brain Res. 2002 Dec 27;958(2):439-47.<br />
<BR><br />
34. http://www.gallup.com/poll/109066/Beer-Back-DoubleDigit-Lead-Over-Wine-Favored-Drink.aspx<br />
</h6> <br />
|}[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:59:32Z<p>Pnguyen: /* HPLC: Calibration Standards */</p>
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[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
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{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
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|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of ''p''-coumaric acid and 20ug/mL of ''trans''-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve.''' Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<BR><br />
<BR><br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C7. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
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[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:59:06Z<p>Pnguyen: /* HPLC: Calibration Standards */</p>
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[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of ''p''-coumaric acid and 20ug/mL of ''trans''-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve.''' Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<BR><br />
<BR><br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C7. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:56:26Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve.''' Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<BR><br />
<BR><br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C7. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/CONCLUSIONSTeam:Rice University/CONCLUSIONS2008-10-30T03:55:13Z<p>Pnguyen: </p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]]<BR><br />
<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
*'''Ongoing work includes:'''<br />
**Characterization of resveratrol production using only 4CL:STS and feeding ''p''-coumaric acid. <br />
**Construction of the full biosynthetic circuit and characterization of resveratrol production.<br />
**Redesign circuit so that an antibiotic selection using bleocin (toxic) is not required. <br />
**Possible switch to fluorescent screen (green beer?)<br />
**Adding woodchips to feed additional coumaric acid into the fermenting mix for additional resveratrol gain and improved taste.<br />
**Exploration of using our biosynthetic circuit in other industrial strains of yeast for resveratrol infused cider, wine, whiskey, bourbon, vodka, etc.<br />
<br />
|}<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/CONCLUSIONSTeam:Rice University/CONCLUSIONS2008-10-30T03:53:46Z<p>Pnguyen: </p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]]<BR><br />
<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
*Ongoing work includes<br />
**Characterization of resveratrol production using only 4CL:STS and feeding ''p''-coumaric acid <br />
**Construction of the full biosynthetic circuit and characterization of resveratrol production<br />
**Redesign circuit so that an antibiotic selection using bleocin (toxic) is not required. <br />
**Possible switch to fluorescent screen (green beer?)<br />
**Adding woodchips to feed additional coumaric acid into the fermenting mix for additional resveratrol gain and improved taste.<br />
**Exploration of using our biosynthetic circuit in other industrial strains of yeast for resveratrol infused cider, wine, whiskey, bourbon, vodka, etc.<br />
<br />
|}<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:51:36Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve. Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<BR><br />
<BR><br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C7. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:51:17Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve. Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C7. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:51:04Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|500px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve. Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:50:52Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|400px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve. Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/File:Wine_graph.jpgFile:Wine graph.jpg2008-10-30T03:50:25Z<p>Pnguyen: </p>
<hr />
<div></div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:49:49Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:Wine_graph.jpg|center|thumb|650px|'''Figure C6: Resveratrol amounts as calculated using the above chromatograms and the calibration curve. Red wine samples are shown in red, white wine samples in yellow. The Chardonnay sample produced no detectable ''trans''-resveratrol peak.]]<br />
<br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:39:54Z<p>Pnguyen: /* HPLC: Calibration Standards */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green). The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol. The inset shows the calibration curve (Peak area vs. Concentration).]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/File:Coumaric_standards.jpgFile:Coumaric standards.jpg2008-10-30T03:38:47Z<p>Pnguyen: uploaded a new version of "Image:Coumaric standards.jpg"</p>
<hr />
<div></div>Pnguyenhttp://2008.igem.org/File:RSV_standards.jpgFile:RSV standards.jpg2008-10-30T03:38:17Z<p>Pnguyen: uploaded a new version of "Image:RSV standards.jpg"</p>
<hr />
<div></div>Pnguyenhttp://2008.igem.org/File:RSV_standards.jpgFile:RSV standards.jpg2008-10-30T03:37:40Z<p>Pnguyen: uploaded a new version of "Image:RSV standards.jpg"</p>
<hr />
<div></div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:20:54Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<BR><BR><br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:20:38Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line).]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:19:32Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line)]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:19:05Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|'''Figure C6. ''beta''-glucosidase treatment of wine samples.''' Shown is a Shiraz wine extract treated with ''beta''-glucosidase (purple) or untreated (red). ]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:17:24Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments indicate a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|700px|Figure ]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/File:Glyco-RSV.jpgFile:Glyco-RSV.jpg2008-10-30T03:15:49Z<p>Pnguyen: </p>
<hr />
<div></div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:15:35Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase increases the amount of resveratrol detected by converting glycosylated resveratrol (aka piceid) into its aglycone form.<br />
:*Our experiments show a marginal increase in ''trans''-resveratrol detection by HPLC after treatment with 500U of beta-glucosidase (Fisher). Other wine varieties may contain a greater percentage of resveratrol in piceid form.<br />
<BR><br />
[[Image:glyco-RSV.jpg|center|thumb|600px]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:11:22Z<p>Pnguyen: /* HPLC: beta-Glucosidase treated samples */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: ''beta''-Glucosidase treated samples=====<br />
:*Some papers show that treatment of wine samples by ''beta''-glucosidase liberates<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:07:59Z<p>Pnguyen: /* C. HPLC Data */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see [[Team:Rice_University/STRATEGY|STRATEGY]]). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: beta-Glucosidase treated samples=====<br />
:*Some papers show that treatment <br />
<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:07:16Z<p>Pnguyen: /* C. HPLC Data */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL will produce resveratrol with the need for supplemented ''p''-coumaric acid (see STRATEGY). Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
===== HPLC: beta-Glucosidase treated samples=====<br />
:*Some papers show that treatment <br />
<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:04:47Z<p>Pnguyen: /* HPLC: Fermenation batches */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<BR><BR><br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:02:40Z<p>Pnguyen: /* HPLC: Calibration */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration Standards=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<BR><br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T03:01:59Z<p>Pnguyen: /* C. HPLC Data */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of ''p''-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of ''trans''-resveratrol and ''p''-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a ''trans''-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: ''trans''-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: ''p''-Coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: ''trans''-resveratrol and ''p''-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain ''trans''-resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a ''trans''-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<BR><br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:59:48Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: trans-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: p-Coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: trans-resveratrol and p-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wines (Fig. C5) contain resveratrol in significant amounts, whereas white wines (Fig. C6) do not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<BR><br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:58:50Z<p>Pnguyen: /* C. HPLC Data */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|'''Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.''']]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|'''Figure C2: trans-resveratrol standards (Sigma).''' Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|'''Figure C3: p-Coumaric acid standards (Sigma).''' Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|'''Figure C4: trans-resveratrol and p-coumaric acid have different retention times.''' This separation will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wine contains resveratrol in significant amounts, whereas white wine does not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: Red wine extracts analyzed by HPLC.''' A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|'''Figure C5: White wine extracts analyzed by HPLC.''' A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<BR><br />
We are currently in the process of transforming our yeast strain; HPLC data of the extracts for this strain will be added soon.<br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:55:17Z<p>Pnguyen: /* D. Fermentation Experiments */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wine contains resveratrol in significant amounts, whereas white wine does not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|Figure C5: White wine extracts analyzed by HPLC. A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|400px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
<BR><br />
More experimental data coming soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:54:41Z<p>Pnguyen: /* D. Fermentation Experiments */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wine contains resveratrol in significant amounts, whereas white wine does not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|Figure C5: White wine extracts analyzed by HPLC. A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg|center|thumb|600px|'''Figure D1. Test beer fermentation using the SAB-hefeweizen brewing strain provided by Saint Arnold Brewery (Houston, TX)'''.]]<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:53:15Z<p>Pnguyen: /* Fermentation */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wine contains resveratrol in significant amounts, whereas white wine does not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|Figure C5: White wine extracts analyzed by HPLC. A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== D. Fermentation Experiments ===<br />
[[Image:Lab17.jpg]]<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:50:42Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
:* The results clearly show that red wine contains resveratrol in significant amounts, whereas white wine does not, as expected.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|650px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|650px|Figure C5: White wine extracts analyzed by HPLC. A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/File:White_Wine_Samples.jpgFile:White Wine Samples.jpg2008-10-30T02:49:27Z<p>Pnguyen: uploaded a new version of "Image:White Wine Samples.jpg"</p>
<hr />
<div></div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:47:05Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|700px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a Merlot from ''Australia'' (red), a Shiraz from ''Australia'' (green), and a Primitivo from ''S. Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|600px|Figure C5: White wine extracts analyzed by HPLC. A)Chromatogram of the white wine extracts; the different wines shown are a Sauvignon Blanc from ''France'' (red) and a Chardonnay from ''N. California'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/File:Red_Wine_Samples.jpgFile:Red Wine Samples.jpg2008-10-30T02:44:13Z<p>Pnguyen: uploaded a new version of "Image:Red Wine Samples.jpg"</p>
<hr />
<div></div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:41:24Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|800px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a merlot from ''Australia'' (red), a shiraz from ''Australia'' (green), and a primitivo from ''Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|600px|Red wine]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:41:03Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|900px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a merlot from ''Australia'' (red), a shiraz from ''Australia'' (green), and a primitivo from ''Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|600px|Red wine]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:40:48Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|925px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a merlot from ''Australia'' (red), a shiraz from ''Australia'' (green), and a primitivo from ''Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|600px|Red wine]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:40:35Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|950px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a merlot from ''Australia'' (red), a shiraz from ''Australia'' (green), and a primitivo from ''Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|600px|Red wine]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:40:12Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|1000px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a merlot from ''Australia'' (red), a shiraz from ''Australia'' (green), and a primitivo from ''Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|600px|Red wine]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:39:14Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
<br />
{| style="color:#1b2c9a;background-color:#FFFFFF;" cellpadding="0" cellspacing="0" border="0" bordercolor="#000" width="100%" align="center"|}<br />
{|align="justify" style="background-color:#FFFFFF;text-align:justify" cellpadding="30" width="90%"<br />
|<br />
=== A. Genetic Engineering and Part Construction===<br />
<br />
A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
<br />
Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
<br />
=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
<br />
=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
<br />
{|align="center" style="background-color:#FFFF99; text-align:left" border="1" cellpadding="0" width="50%"<br />
|<br />
:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
|}<BR><br />
[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
<BR><br />
===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
<br />
[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
<br />
[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
<br />
===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
<BR><br />
[[Image:Red_Wine_Samples.jpg|center|thumb|900px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a merlot from ''Australia'' (red), a shiraz from ''Australia'' (green), and a primitivo from ''Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
<BR><br />
[[Image:White_Wine_Samples.jpg|center|thumb|600px|Red wine]]<br />
<br />
===== HPLC: Fermenation batches=====<br />
<br />
=== Fermentation ===<br />
<br />
Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
<br />
|}<br />
<br />
<br />
[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
<br />
|}</div>Pnguyenhttp://2008.igem.org/Team:Rice_University/RESULTSTeam:Rice University/RESULTS2008-10-30T02:38:56Z<p>Pnguyen: /* HPLC: Wine Extract Tests */</p>
<hr />
<div><!--MAIN PROJECT PAGE--><br />
{|<br />
|align="center" style="background:#000080"|<BR><br />
[[Image:rice.jpg|650px|justify]]<br />
[[Image:rice logo.jpg|298px|justify]] <br />
<BR>[[Image:GradientBar.jpg|960px|center]]<br />
|}<br />
{|<br />
|align="center" style="background:#C0C0C0"| <BR><br />
[[Image:ProjectTitle.jpg]] <BR><br />
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[[Team:Rice_University/OUR TEAM|OUR TEAM]]:::[[Team:Rice_University|SUMMARY]] ::: [[Team:Rice_University/BACKGROUND|BACKGROUND]] ::: <br />
[[Team:Rice_University/STRATEGY|STRATEGY]] ::: [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] ::: [[Team:Rice_University/RESULTS|RESULTS]] ::: [[Team:Rice_University/CONCLUSIONS|ONGOING WORK]]<br />
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=== A. Genetic Engineering and Part Construction===<br />
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A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the [[Team:Rice_University/CONSTRUCTS|CONSTRUCTS]] page.<br />
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Please visit our [[Team:Rice_University/NOTEBOOK|Notebook]] for a summary of labwork and protocols.<BR><BR><br />
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=== B. Yeast Transformation ===<br />
We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon. <BR><BR><br />
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=== C. HPLC Data===<br />
To analyze our beer samples for resveratrol content, be will be using '''H'''igh '''P'''erformance '''L'''iquid '''C'''hromatography (HPLC), which will allow us to separate the metabolites produced by the yeast and analyze these compounds by spectrophotometry. By comparing HPLC chromatogram peaks of metabolites produced by our yeast with a resveratrol-only standard, we can identify if resveratrol is being produced, and at what quantities. We will be monitoring the production of resveratrol and the consumption of p-coumaric acid, which will only be added to the media for the 4CL::STS-integrated yeast; the 4CL::STS+TAL . Below, we show our initial data for HPLC calibration curves using known quantities of resveratrol and p-coumaric acid standards and test chromatograms using extracts from different wine samples.<br />
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:'''HPLC Parameters'''<BR><br />
::''Instrument'': Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.<br />
::''Column'': Agilent Eclipse XDB-C18, 5uM (9.4x250mm)<BR><br />
::''Mobile Phases'': <br />
::::(A) 5% acetonitrile / 0.95% acetic acid<br />
::::(B) 70% acetonitrile / 0.3% acetic acid<br />
::''Linear gradient'': A to B over 29 minutes<br />
::''Flow rate'': 0.9mL/min<br />
::''Absorbance monitoring'': 290nm<br />
::''Sample injection volume'': 25 microliters<br />
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[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]<br />
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===== HPLC: Calibration=====<br />
[[Image:RSV_standards.jpg|center|thumb|600px|Figure C2: trans-resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL (red), 10ug/mL (purple), 5ug/mL (blue), 2.5ug/mL (green).]]<BR><br />
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[[Image:Coumaric_standards.jpg|center|thumb|600px|Figure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL (red), 9.25ug/mL (purple), 4.63ug/mL (blue), 2.3ug/mL (green).]]<BR><br />
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[[Image:RSVandCoumaric.jpg|center|thumb|600px|Figure C4: trans-resveratrol and p-coumaric acid separate at different times; this will allow us to resolve both molecules in a complex mixture, such as a yeast extract. Shown is 18.5ug/mL of p-coumaric acid and 20ug/mL of trans-resveratrol.]]<BR><br />
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===== HPLC: Wine Extract Tests=====<br />
:* All wine samples were extracted using 6 volumes of ethyl acetate. The ethyl acetate extracts were then evaporated under vacuum and then resuspended with 70% methanol. Samples were kept in light-protected tubes until analysis.<br />
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[[Image:Red_Wine_Samples.jpg|center|thumb|800px|Figure C5: Red wine extracts analyzed by HPLC. A)Chromatogram of the red wine extracts; the different wines shown are a merlot from ''Australia'' (red), a shiraz from ''Australia'' (green), and a primitivo from ''Italy'' (purple). Also shown is a trans-resveratrol standard (10ug/mL, blue dotted line). B)Zoomed-in view of the resveratrol peak region.]]<br />
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[[Image:White_Wine_Samples.jpg|center|thumb|600px|Red wine]]<br />
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===== HPLC: Fermenation batches=====<br />
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=== Fermentation ===<br />
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Coming Soon. For a sneak preview, check out the [[Team:Rice_University/GALLERY|Gallery]]<br />
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[[Team:Rice_University/OUR TEAM|OUR TEAM]] ::: <br />
[[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]] <br />
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|}</div>Pnguyen