Team:TU Delft/Project/rbs-characterization/results

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(New page: The RBS strength defines how much of a protein is produced compared to a reference RBS sequence. However, RBS characterization measurements only include current protein level (GFP measurem...)
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The RBS strength defines how much of a protein is produced compared to a reference RBS sequence. However, RBS characterization measurements only include current protein level (GFP measurements) and current biomass concentration (Absorption measurements). During measurements, the protein concentration is also influenced by other factors:
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__NOTOC__
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* The rapid growth of the bacteria will dilute the protein considerably
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{{Team:TU_Delft/frame_check}}
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* Proteins degrade over time.
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#rbs-results table {
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#rbs-results table td {
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From the biomass, or optical density (OD), graph below, it can be seen that the fastest growth occurs from 50 minutes until about 3 hours into the experiment. Within this timespan, it is assumed that growth is exponential, and growth rate can be calculated.  
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#rbs-results table .head {
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<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html>
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==The Results==
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In the plot below the RBS strengths for the five tested members of the Anderson family are given along with the deviations from the mean. The strengths are given relative to the community RBS [http://partsregistry.org/Part:BBa_B0034 BBa_B0034], which has a strength of unity (1). This relationship could be built by our characterization of the community RBS [http://partsregistry.org/Part:BBa_B0032 BBa_B0032] simultaneously, which is known to have a strength (or efficiency) of 0.3 relative to [http://partsregistry.org/Part:BBa_B0032 BBa_B0034]. Therefore in the plot below, the mean value of BBa_B0032 is set to 0.3. The values have been obtained by the experimental setup described [[Team:TU_Delft/Project/rbs-characterization/parts|here]] and using the [[Team:TU_Delft/Modeling/protein-production-model|model]] to correct for growth dilution.
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[[Image:TUDelft_2010_RBS_strength_graph.PNG|600px|thumb|center|'''Figure 1.''' RBS strength]]
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<html><div id="rbs-results"></html>
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[[Image:TUDelft2010_Rbs_strength_boxplot.png]]
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==Conclusions==
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The given relative strengths are displayed assuming B0034 as the unit.
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We've succeeded in characterizing five members of the Anderson ribosome binding site family by 96 well plate cultivation and simultaneous monitoring of GFP fluorescence and biomass absorbance. Fitting the obtained data to our improved [https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization protein production model] yielded RBS strengths values with negligible standard deviations. To recap the following RBS strengths (efficiencies) were found:
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{|border="1"
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|RBS
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<html><!-- Original data
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|Mean strength
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J61100 0.019970|0.005119
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|Standard deviation
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J61101 0.119333|0.021401
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J61107 0.077024|0.014804
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J61117 0.012552|0.004484
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J61127 0.065226|0.006604
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B0032 0.300000|0.026904
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Rounded, in percentage
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J61100 0.020|0.00512
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J61101 0.119|0.02140
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J61107 0.077|0.01480
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J61117 0.013|0.00448
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J61127 0.065|0.00660
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B0032 0.300|0.02690
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--></html>
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{|
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|<b>Ribosome binding site</b>
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|<b>Mean relative strength</b>
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|<b>Standard deviation</b>
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|-
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|J61100
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|[http://partsregistry.org/Part:BBa_J61100 J61100]
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|0.026560
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|0.020 (2.0%)
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|0.005126
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|0.00512
|-
|-
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|J61117
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|[http://partsregistry.org/Part:BBa_J61101 J61101]
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|0.123819
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|0.119 (11.9%)
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|0.024725
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|0.02140
|-
|-
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|J61101
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|[http://partsregistry.org/Part:BBa_J61107 J61107]
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|0.081210
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|0.077 (7.7%)
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|0.013402
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|0.01480
|-
|-
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|J61107
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|[http://partsregistry.org/Part:BBa_J61117 J61117]
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|0.019763
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|0.013 (1.3%)
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|0.003963
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|0.00448
|-
|-
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|J61127  
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|[http://partsregistry.org/Part:BBa_J61127 J61127]
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|0.069657
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|0.065 (6.5%)
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|0.007422
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|0.00660
|-
|-
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|B0032
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|[http://partsregistry.org/Part:BBa_B0032 B0032]
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|0.300000
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|0.300 (Reference, 30%)
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|0.028316
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|0.02690
|}
|}
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<html></div></html>
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<html><center><img src="https://static.igem.org/mediawiki/2010/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="https://2010.igem.org/Team:TU_Delft#page=Project/rbs-characterization/parts" target="" /></map></html>

Latest revision as of 21:28, 27 October 2010

CharacterizationResultsParts

The Results

In the plot below the RBS strengths for the five tested members of the Anderson family are given along with the deviations from the mean. The strengths are given relative to the community RBS [http://partsregistry.org/Part:BBa_B0034 BBa_B0034], which has a strength of unity (1). This relationship could be built by our characterization of the community RBS [http://partsregistry.org/Part:BBa_B0032 BBa_B0032] simultaneously, which is known to have a strength (or efficiency) of 0.3 relative to [http://partsregistry.org/Part:BBa_B0032 BBa_B0034]. Therefore in the plot below, the mean value of BBa_B0032 is set to 0.3. The values have been obtained by the experimental setup described here and using the model to correct for growth dilution.

Figure 1. RBS strength

Conclusions

We've succeeded in characterizing five members of the Anderson ribosome binding site family by 96 well plate cultivation and simultaneous monitoring of GFP fluorescence and biomass absorbance. Fitting the obtained data to our improved protein production model yielded RBS strengths values with negligible standard deviations. To recap the following RBS strengths (efficiencies) were found:


Ribosome binding site Mean relative strength Standard deviation
[http://partsregistry.org/Part:BBa_J61100 J61100] 0.020 (2.0%) 0.00512
[http://partsregistry.org/Part:BBa_J61101 J61101] 0.119 (11.9%) 0.02140
[http://partsregistry.org/Part:BBa_J61107 J61107] 0.077 (7.7%) 0.01480
[http://partsregistry.org/Part:BBa_J61117 J61117] 0.013 (1.3%) 0.00448
[http://partsregistry.org/Part:BBa_J61127 J61127] 0.065 (6.5%) 0.00660
[http://partsregistry.org/Part:BBa_B0032 B0032] 0.300 (Reference, 30%) 0.02690

CharacterizationResultsParts

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