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

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(New page: =Characterization= <html><div style='clear:both'> The cells were cultured over 18 hours in 96-well plates using a Gen5 fluorescence and absorbance plate reader, LB with ampicillin was the ...)
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=Characterization=
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<html><div style='clear:both'> The cells were cultured over 18 hours in 96-well plates using a Gen5 fluorescence and absorbance plate reader, LB with ampicillin was the culture medium used in these experiments. Only the results obtained during exponential growth phase were taken into account.  
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{{Team:TU_Delft/frame_check}}
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</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>
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[[Image:26_07_2010_Rbs.png|center]]
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==Characterization==
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'''Note:''' Fluorescence (y-axis) is reported as Arbitrary Fluorescence Units.
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<i>By varying only the RBS sequence in a protein generator construct the dependence of the protein expression level on the RBS sequence could be determined by simple fluorescence and biomass curve analysis. The methods are further explained below.</i>
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The RBS strength was calculated by taking the mean of the ratio between the expression of the standard RBS ([http://partsregistry.org/Part:BBa_B0032 B0032]) and expression of Anderson RBS over time. The Expression is defined as the quotient of the measured fluorescence divided by measured biomass (OD at 600nm).
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=== What is RBS strength, really ? ===
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In the field of synthetic biology the 'strength' of an RBS is defined as the rate at which translation is initiated by the RBS sequence on any given mRNA molecule. Going back a step, we see that translation initiation occurs preferentially at certain mRNA sequences, which show a similarity to the consensus -Shine-Delgarno- sequence. This is due to the optimal binding of the 16S rRNA at these regions. In other words, the RBS 'strength' may loosely be defined as the rate of ribosome binding to any given mRNA molecule. (Click [http://partsregistry.org/Help:Ribosome_Binding_Sites/Mechanism here] for more information.)
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" align="center"
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For our project it is not directly the binding equilibria of ribosome to mRNA that is of interest to us, but rather the netto rate of protein production. Thus, in our experimentation we define the RBS strength as the production rate of a given protein downstream of an RBS.
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|'''RBS'''
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|'''Strength'''
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|[http://partsregistry.org/Part:BBa_J61100 J61100]
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|0.047513
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|[http://partsregistry.org/Part:BBa_J61101 J61101]
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|0.119831
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|-
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|[http://partsregistry.org/Part:BBa_J61107 J61107]
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|0.065454
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|-
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|[http://partsregistry.org/Part:BBa_J61117 J61117]
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|0.038518
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|[http://partsregistry.org/Part:BBa_J61127 J61127]
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|0.087334
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|[http://partsregistry.org/Part:BBa_B0032 B0032]
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|0.300000
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|}
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The result of this experimental set up was a simple characterization of five of the Anderson RBS sequences in relation to other standard well-characterized RBS. The given relative strengths are displayed assuming [http://partsregistry.org/Part:BBa_B0034 B0034] as the unit.
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==The experiment==
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The <i>E.coli strains</i> carrying the parts to be tested were cultured for 16 hours on 96-well plates while measuring fluorescence of GFP and biomass absorbance at 10 minute intervals. Click [https://2010.igem.org/Team:TU_Delft/Protocols#RBS_characterization_experiment here] for the more detailed protocol.
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We're also trying to look into mRNA folded shapes using mfold to see if there is a common pattern in the Anderson RBS shapes. This might be usable in predicting RBS strength for the other untested Anderson RBS sequences. Unfortunately, this seems not to be the case, as all the RBS sequences have very different mfold shapes.
 
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=== Data normalization method ===
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To go from the raw measurements to the RBS strengths, we ran the following steps using a matlab script:
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== Source data ==
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[[Image:TUDelft2010_Rbs-control-gfp.png|right|thumb|300px]]
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the plate reader data consists of 8 rows, each containing multiples of 12:
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* 5 strains with Anderson RBSs to be characterized
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* B0032 strain to compare to
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* I13401 strain as control (GFP-dT without promotor and RBS)
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* LB+Amp as blank
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Source code and data used for characterization:
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To prepare the data for further calculation, the following was done:
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* Subtract the blank GFP and OD values from the data of wells containing E coli. This takes care of any background noise caused by the LB medium.
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* The strain containing I13401 produces some nonzero measurements (See right). From this control strain measurements we can calculate how much autofluorescence is reported due to biomass or due to leaky expression.
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* For each well, we calculate the GFP that is not a result of biomass or leaky expression:
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[[Image:TUDelft_2010_PPM_Autofluorescence.png]]
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* These values are curve fitted into our [[Team:TU_Delft/Modeling/protein-production-model|protein production model]], described in the In Silico section. This results in a (exponential phase) protein production rate for each plate reader well. These production rates are then averaged and compared to the B0032 reference RBS to produce our [[Team:TU_Delft/Project/rbs-characterization/results|resulting RBS strengths]].
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Data: [https://static.igem.org/mediawiki/2010/b/bf/Experiment1_26_7_10.m Experiment1_26_7_10.m]
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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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Matlab calculation code: [https://static.igem.org/mediawiki/2010/5/5d/IGEM_TUDelft_2010_Rbs_calc.m IGEM_TUDelft_2010_Rbs_calc.m]
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Latest revision as of 19:47, 27 October 2010

CharacterizationResultsParts

Characterization

By varying only the RBS sequence in a protein generator construct the dependence of the protein expression level on the RBS sequence could be determined by simple fluorescence and biomass curve analysis. The methods are further explained below.

What is RBS strength, really ?

In the field of synthetic biology the 'strength' of an RBS is defined as the rate at which translation is initiated by the RBS sequence on any given mRNA molecule. Going back a step, we see that translation initiation occurs preferentially at certain mRNA sequences, which show a similarity to the consensus -Shine-Delgarno- sequence. This is due to the optimal binding of the 16S rRNA at these regions. In other words, the RBS 'strength' may loosely be defined as the rate of ribosome binding to any given mRNA molecule. (Click here for more information.)

For our project it is not directly the binding equilibria of ribosome to mRNA that is of interest to us, but rather the netto rate of protein production. Thus, in our experimentation we define the RBS strength as the production rate of a given protein downstream of an RBS.

The experiment

The E.coli strains carrying the parts to be tested were cultured for 16 hours on 96-well plates while measuring fluorescence of GFP and biomass absorbance at 10 minute intervals. Click here for the more detailed protocol.


Data normalization method

To go from the raw measurements to the RBS strengths, we ran the following steps using a matlab script:

TUDelft2010 Rbs-control-gfp.png

the plate reader data consists of 8 rows, each containing multiples of 12:

  • 5 strains with Anderson RBSs to be characterized
  • B0032 strain to compare to
  • I13401 strain as control (GFP-dT without promotor and RBS)
  • LB+Amp as blank

To prepare the data for further calculation, the following was done:

  • Subtract the blank GFP and OD values from the data of wells containing E coli. This takes care of any background noise caused by the LB medium.
  • The strain containing I13401 produces some nonzero measurements (See right). From this control strain measurements we can calculate how much autofluorescence is reported due to biomass or due to leaky expression.
  • For each well, we calculate the GFP that is not a result of biomass or leaky expression:

TUDelft 2010 PPM Autofluorescence.png

  • These values are curve fitted into our protein production model, described in the In Silico section. This results in a (exponential phase) protein production rate for each plate reader well. These production rates are then averaged and compared to the B0032 reference RBS to produce our resulting RBS strengths.

CharacterizationResultsParts

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