Team:Warsaw/RBSmeasurement

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<h2>Project description</h2>
 
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<!-- <div class="note">Here goes something about our project</div> -->
 
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<div class="note">RBS Measurement</div>
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<div class="note">RBS Measurement</div><br/>
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<img src="https://static.igem.org/mediawiki/2011/f/f2/Tabelka.png" alt="ERROR" align="left" />
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<b>General info</b><p align=justify> We have conducted measurement of various ribosome binding sites included in 2011 spring distribution. Our list of measured parts includes YFP E0030 , GFP SUPERFOLDER , RFP E1010, mOrange E2050, GFP  E0040. DNA of SUPERFOLDER GFP, was included in the distribution as a part of a bigger biobrick. We obtained coding sequence of SUPERFOLDER GFP using PCR method. All measurements were conducted in psb1A2 - a high copy number vector that was used in previous RBS measurement experiments with GFP protein.</p>
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In order to fine-tune expression of genes used in our project we have conducted measurement of various ribosome binding sites included in 2010 spring distribution. Our list of measured parts includes RBSs both from Community and Anderson's collections. We have used standard measurement kit composed of promoter BBa_J23100 and GFP+terminator part BBa_I130401. All measurements were carried out using flow cytometer.<br />
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<b>Sample preparation</b><p align="justify">
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<b>Sample preparation</b><br />
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To measure fluorescence of samples 3 milliliters of LB with AMP were inoculated with a colony from the plate. Cultures were vigorously shaken in 37° C overnight. The next step was to centrifuge 1,5 milliliters of liquid culture in an 1,5 mltube. Pellet was resuspended in 100 microliters of RF. Finally, prepared samples were placed in the
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To measure fluorescence of samples 3 milliliters of LB were inoculated with a colony from the plate. Cultures were vigorously shaken in 37° C overnight. The next step was to centrifuge 1,5 milliliters of liquid culture in an eppendorf tube. Pellet was then resuspended in 100 microliters of RF. Finally, prepared samples were applied on the
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fluorimeter plate. We used Corning 96 deep well plates with clear UV transparent bottom. Measurements were carried out using Tecan HS2000 pro fluorimeter with bottom read mode and using active gain optimization
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fluorimeter plates. We used Corning 96 deep well plates with clear UV transparent bottom. Measurments were carried out using Tecan HS2000 pro fluorimeter with bottom read mode and using active gain optimization
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function. Wavelength values for individual proteins:</p><br />
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function. Wavlength values for individual proteins:
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<th></th><th>GFP</th><th>YFP</th><th>RFP</th><th>mORANGE</th><th>SF-GFP</th>
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<td>Excitation [nm]</td><td>488</td><td>514</td><td>554</td><td>545</td><td>488</td>
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<td>Emission [nm]</td><td>509</td><td>530</td><td>585</td><td>568</td><td>508</td>
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We also conducted flow cytometry measurements for majority of our parts.
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<dt>GFP</dt>
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<dd>Excitation 488 nm</dd>
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<dd>Emission 509 nm</dd>
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<dt>YFP</dt>
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<dd>Excitation 514 nm</dd>
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<dd>Emission 530 nm</dd>
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<dt>RFP</dt>
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<dd>Excitation 554 nm</dd>
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<dd>Emission 585 nm</dd>
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<dt>OFP</dt>
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<dd>Excitation 545 nm</dd>
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<dd>Emission 568 nm</dd>
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<dt>SF-GFP</dt>
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<dd>Excitation 488 nm</dd>
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<dd>Emission 508 nm</dd>
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Latest revision as of 22:42, 21 September 2011

Example Tabs


RBS Measurement

General info

We have conducted measurement of various ribosome binding sites included in 2011 spring distribution. Our list of measured parts includes YFP E0030 , GFP SUPERFOLDER , RFP E1010, mOrange E2050, GFP E0040. DNA of SUPERFOLDER GFP, was included in the distribution as a part of a bigger biobrick. We obtained coding sequence of SUPERFOLDER GFP using PCR method. All measurements were conducted in psb1A2 - a high copy number vector that was used in previous RBS measurement experiments with GFP protein.

Sample preparation

To measure fluorescence of samples 3 milliliters of LB with AMP were inoculated with a colony from the plate. Cultures were vigorously shaken in 37° C overnight. The next step was to centrifuge 1,5 milliliters of liquid culture in an 1,5 mltube. Pellet was resuspended in 100 microliters of RF. Finally, prepared samples were placed in the fluorimeter plate. We used Corning 96 deep well plates with clear UV transparent bottom. Measurements were carried out using Tecan HS2000 pro fluorimeter with bottom read mode and using active gain optimization function. Wavelength values for individual proteins:


GFPYFPRFPmORANGESF-GFP
Excitation [nm]488514554545488
Emission [nm]509530585568508
We also conducted flow cytometry measurements for majority of our parts.

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