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

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==Characterization==
==Characterization==
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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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=== RBS strength ===
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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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* The rapid growth of the bacteria will dilute the protein considerably
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* Proteins degrade over time.
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=== Known methods ===
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=== What is RBS strength, really ? ===
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The conventional method for characterization of RBS or promotor sequences is to assume that the derivative of GFP concentration is the production rate, as shown in the formula from Kelly et al (shown below).
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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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[[Image:TUDelft2010_RBS_1754-1611-3-4-i9.gif]]
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This method only works if cells are subcultured a few times in the exponential phase. In that case, steady state GFP concentration can be assumed and dilution can be ignored.
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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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In our experiment however, this was not the case. Specific GFP is actually decreasing because of dilution, so it needs to be modeled in order to make any use of the data.  
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=== Growth curve fitting ===
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==The experiment==
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From the biomass, or optical density (OD), graph below, it can be seen that the fastest growth occurs from 1 hour until about 3 hours into the experiment. Within this timespan, it is assumed that growth is exponential, and growth rate can be calculated. The graph below shows 72 growth curves. 12 seperate wells were used for every RBS sequence.
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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 [http://2010.igem.org/Team:TU_Delft/Protocols#RBS_characterization_experiment here] for the more detailed protocol.
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[[Image:Tud2010_RBS_OD.png]]
 
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The exponential part of this graph is curve-fitted to an exponential growth model:
 
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[[Image:TUD2010_RBS_Growth_model.PNG]]
 
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Which results in a growth rate ('mu') that can be used to add dilution to further calculation.
 
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=== Biomass growth will dilute the GFP ===
 
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[[Image:TUD2010_Gfpod_all.png|right|400px]]
 
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At the start of our measurements, the GFP protein has reached a steady state high concentration.
 
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The specific GFP will decrease during growth because of dilution, and this needs to be accounted for when calculating the RBS strength.
 
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<br style="clear: both" />
 
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== Protein production model ==
 
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As said, the protein production model needs to take dilution into account. The GFP protein that is used is very stable (half life 41 hours), so the degradation term is negligible compared to dilution.
 
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[[Image:RBS_Expression_model.PNG]]
 
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In above formula:
 
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* Y is the GFP concentration
 
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* Beta is the production rate (The constant that we are interested in)
 
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* Alpha is the dilution+degradation rate
 
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Converting this equation to an explicit form results in:
 
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[[Image:TU2010_Gfp_explicit.PNG]]
 
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The 2 unknown parameters (beta and Y1) where curve-fitted against the GFP/OD data, using the matlab lsqcurvefit command.
 
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The beta constant for each of the 72 experiments were then to calculate RBS strengths, listed in the  [[Team:TU_Delft/Project/rbs-characterization/results|results]] page.
 
=== Data normalization method ===
=== Data normalization method ===
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To go from original measurements to RBS strenghts, we ran the following steps using a matlab script:
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To go from the raw measurements to the RBS strengths, we ran the following steps using a matlab script:
[[Image:TUDelft2010_Rbs-control-gfp.png|right|thumb|300px]]
[[Image:TUDelft2010_Rbs-control-gfp.png|right|thumb|300px]]
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Our plate reader data consists of 12 columns of
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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 RBS'es to be characterized
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* 5 strains with Anderson RBSs to be characterized
* B0032 strain to compare to
* B0032 strain to compare to
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* I13401 strain as control (GFP without promotor and RBS)
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* I13401 strain as control (GFP-dT without promotor and RBS)
* LB+Amp as blank
* LB+Amp as blank
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To prepare the data for further calculation, some things have to be done:
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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 with E coli in it. This takes care of the platereader error independent of biomass
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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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* Biomass without GFP also produces some nonzero measurements (See right). From the control strain measurements we can calculate how much 'fake' GFP is reported due to biomass.  
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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 real GFP that is not a result of biomass:
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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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real_gfp = gfp - fake_gfp_per_od * od
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[[Image:TUDelft_2010_PPM_Autofluorescence.png]]
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* These values are curve fitted into our protein production model, resulting in a (exponential phase) protein production rate for each well.
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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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=== Source code and data ===
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http://2010.igem.org/wiki/images/8/88/TUDelft_2010_RBS_characterization.zip
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=== References ===
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;Kelly 2009
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:Jason R. Kelly, Adam J. Rubin, Joseph H. Davis, ''et al'' (March 2009). "[http://dx.doi.org/10.1186/1754-1611-3-4 Measuring the activity of BioBrick promoters using an in vivo reference standard]". ''Journal of Biological Engineering'' '''3''': 4
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===Continue Reading about the RBS Characterization===
 
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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