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Team:Warsaw/Stage1/RBSMeas
From 2010.igem.org
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<h2>RBS measurement</h2> | <h2>RBS measurement</h2> | ||
| - | + | <div class="note">Measured parts</div><br> | |
| - | <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_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> | <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_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">Measurement constructs</div> | + | <div class="note">Measurement constructs</div><br> |
<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> | <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> | </html><partinfo>BBa_K299007 DeepComponents</partinfo><html> | ||
| - | <p>All measurements were conducted in <i>E.coli</i> Top10 chassis.</p> | + | <p>All measurements were conducted in <i>E.coli</i> Top10 chassis.</p><br> |
| - | <div class="note">Methodology</div> | + | <div class="note">Methodology</div><br> |
<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 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> | ||
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<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>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> | <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> | + | </ol><br> |
| - | <div class="note">Results and conclusion.</div> | + | <div class="note">Results and conclusion.</div><br> |
| - | <p>The results that we obtained are summarized in the figure below. You can easily notice that all RBS sequences from the Anderson's library are rather weak. Our measurements of community | + | <p>The results that we obtained are summarized in the figure below. You can easily notice that all RBS sequences from the Anderson's library are rather weak. Our measurements of community collection are in agreement with previous experiments, except B0030. In our hands B0030 is stronger than previously measured. It was suppouse to be around 0.6 of B0034, we observed 0.9 of B0034. For detailed comparison of our results with registry and predictions see <a href="https://2010.igem.org/Team:Warsaw/Stage1/Modeling">modeling</a> page.<br> <br> |
| - | We sequenced all measured parts and | + | We sequenced all measured parts and they contained no mutations, also we measured the RBSes independently 2 times, each time all constructs were measured in triplicate. We are really curios if B0030 is actually stronger that we thought or it was our particular measurement system or technique that influenced the results. |
We would like to encourage other iGEM team to re-measure B0030!</p> | We would like to encourage other iGEM team to re-measure B0030!</p> | ||
<center><img src="https://static.igem.org/mediawiki/2010/e/e9/RBS_relative_strength.png"></center> | <center><img src="https://static.igem.org/mediawiki/2010/e/e9/RBS_relative_strength.png"></center> | ||
Latest revision as of 11:04, 26 October 2010
RBS measurement
Measured parts
We have measured following Ribosome Binding Sites from Anderson's collection: J61100 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 Top10 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 experiment 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 and conclusion.
The results that we obtained are summarized in the figure below. You can easily notice that all RBS sequences from the Anderson's library are rather weak. Our measurements of community collection are in agreement with previous experiments, except B0030. In our hands B0030 is stronger than previously measured. It was suppouse to be around 0.6 of B0034, we observed 0.9 of B0034. For detailed comparison of our results with registry and predictions see modeling page.
We sequenced all measured parts and they contained no mutations, also we measured the RBSes independently 2 times, each time all constructs were measured in triplicate. We are really curios if B0030 is actually stronger that we thought or it was our particular measurement system or technique that influenced the results.
We would like to encourage other iGEM team to re-measure B0030!
