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Team:Warsaw/Stage1/PromMeas
From 2010.igem.org
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| - | <div class="note"> | + | <h3>Experimental setup</h3> |
| + | <div class="note">Measured parts</div> | ||
| + | <p>We have measured following Ribosome Binding Sites from Anderson's collection: <a href="http://partsregistry.org/Part:BBa_J61100">J61100</a> <a href="http://partsregistry.org/Part:BBa_J61101">J61101</a> <a href="http://partsregistry.org/Part:BBa_J61107">J61107</a> <a href="http://partsregistry.org/Part:BBa_J61117">J61117</a> <a href="http://partsregistry.org/Part:BBa_J61127">J61127</a>. Additionally we have measured following parts from the Community collection: <a href="http://partsregistry.org/Part:BBa_B0030">B0030</a> <a href="http://partsregistry.org/Part:BBa_B0031">B0031</a> <a href="http://partsregistry.org/Part:BBa_B0032">B0032</a> <a href="http://partsregistry.org/Part:BBa_B0033">B0033</a> <a href="http://partsregistry.org/Part:BBa_B0034">B0034</a></p><br> | ||
| - | <div class="note"> | + | <div class="note">Measurement constructs</div> |
| + | <p>We have used <a href="http://partsregistry.org/Part:BBa_J23100">J23100</a> as our reference promoter and <a href="http://partsregistry.org/Part:BBa_E0040">E0040</a> GFP as a reporter. <a href="http://partsregistry.org/Part:BBa_K299007">Example construct</a>:</p> | ||
| + | </html><partinfo>BBa_K299007 DeepComponents</partinfo><html> | ||
| + | <p>All measurements were conducted in E. coli Tpo10 chassis.</p> | ||
| + | <div class="note">Methodology</div> | ||
| + | <p>We have decided to use Fluorescence Activated Cell Sorter (aka. FACS) as our measurement device. This allowed us to achieve greater precision because we didn't have to determine both OD and fluorescence separately (accumulated error from 2 measurements). Also flow cytometry allowed us to measure large population of cells in single run. You can think about this as conducting 30 000 independent experiments at once.</p> | ||
| + | <p> We have used following protocol:</p> | ||
| + | <ol> | ||
| + | <li>Each expreiment was started from the fresh transformation of Top10 cells</li> | ||
| + | <li>Strains encoding our measurement constructs were seeded from single colony and cultured in LB broth overnight to reach saturation phase. Measurement of each RBS was preformed from three independent cultures.</li> | ||
| + | <li>Overnight cultures were spun down, resuspended in equal amount of PBS supplemented with 500mg/ml kanamycin and measured on BD FacsCalibur flow cytometer.</li> | ||
| + | </ol> | ||
| + | |||
| + | <h3>Results</h3> | ||
| + | <p>We have obtained following results:</p> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2010/e/e9/RBS_relative_strength.png"></center> | ||
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Revision as of 21:41, 16 October 2010
Promoter measurement
Experimental setup
Measured parts
We have measured following Ribosome Binding Sites from Anderson's collection: J61100 J61101 J61107 J61117 J61127. Additionally we have measured following parts from the Community collection: B0030 B0031 B0032 B0033 B0034
Measurement constructs
We have used J23100 as our reference promoter and E0040 GFP as a reporter. Example construct:
<partinfo>BBa_K299007 DeepComponents</partinfo>All measurements were conducted in E. coli Tpo10 chassis.
Methodology
We have decided to use Fluorescence Activated Cell Sorter (aka. FACS) as our measurement device. This allowed us to achieve greater precision because we didn't have to determine both OD and fluorescence separately (accumulated error from 2 measurements). Also flow cytometry allowed us to measure large population of cells in single run. You can think about this as conducting 30 000 independent experiments at once.
We have used following protocol:
- Each expreiment was started from the fresh transformation of Top10 cells
- Strains encoding our measurement constructs were seeded from single colony and cultured in LB broth overnight to reach saturation phase. Measurement of each RBS was preformed from three independent cultures.
- Overnight cultures were spun down, resuspended in equal amount of PBS supplemented with 500mg/ml kanamycin and measured on BD FacsCalibur flow cytometer.
Results
We have obtained following results:
