Team:Rice University/RESULTS

From 2008.igem.org

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=== C. HPLC Data===
=== C. HPLC Data===
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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. 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.
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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.
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::''Sample injection volume'': 25 microliters
::''Sample injection volume'': 25 microliters
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[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a resveratrol standard.]]
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[[Image:HPLC_RSV_example.jpg|center|thumb|600px|Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.]]
===== HPLC: Calibration=====
===== HPLC: Calibration=====
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[[Image:RSV_standards.jpg|center|thumbs|600px|Figure C2: Resveratrol standards (Sigma). Different serial dilutions are shown: 20 ug/mL, 10ug/mL, 5ug/mL, 2.5ug/mL]]
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[[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>
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[[Image:Coumaric_standards.jpg|center|thumbs|600pxFigure C3: p-Coumaric acid standards (Sigma). Different serial dilutions are shown: 18.5ug/mL, 9.25ug/mL, 4.63ug/mL, 2.3ug/mL]]
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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>
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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.]]<BR>]]
===== HPLC: Wine Extract Tests=====
===== HPLC: Wine Extract Tests=====

Revision as of 02:25, 30 October 2008


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

Contents

A. Genetic Engineering and Part Construction

A detailed summary of parts, design considerations, and genetic engineering progress can be accessed at the CONSTRUCTS page.

Please visit our Notebook for a summary of labwork and protocols.

B. Yeast Transformation

We are currently accumulating data for plasmid transformations into our brewing strain, SAB-Hefe. Data will be updated soon.

C. HPLC Data

To analyze our beer samples for resveratrol content, be will be using High Performance Liquid Chromatography (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.

HPLC Parameters
Instrument: Shimadzu LC-10AT liquid chromatography unit, SPD-10A UV-vis detector, and SCL-10A system controller.
Column: Agilent Eclipse XDB-C18, 5uM (9.4x250mm)
Mobile Phases:
(A) 5% acetonitrile / 0.95% acetic acid
(B) 70% acetonitrile / 0.3% acetic acid
Linear gradient: A to B over 29 minutes
Flow rate: 0.9mL/min
Absorbance monitoring: 290nm
Sample injection volume: 25 microliters
Figure C1. Example HPLC chromatogram of a trans-resveratrol standard.
HPLC: Calibration
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).

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).

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.

]]
HPLC: Wine Extract Tests
HPLC: Fermenation batches

Fermentation

Coming Soon. For a sneak preview, check out the Gallery


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

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