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

From 2010.igem.org

(Difference between revisions)
(→Data normalization method)
 
(72 intermediate revisions not shown)
Line 1: Line 1:
-
===Characterization===
+
__NOTOC__
 +
{{Team:TU_Delft/frame_check}}
 +
<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>
 +
==Characterization==
 +
<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>
-
== Plots of measurements ==
+
=== What is RBS strength, really ? ===
-
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:
+
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.)
-
* The rapid growth of the bacteria will dilute the protein considerably
+
-
* Proteins degrade over time.
+
-
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. The graph below shows 72 growth curves. 12 seperate wells were used for every RBS sequence.
+
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.
-
[[Image:Tud2010_RBS_OD.png]]
+
==The experiment==
 +
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.
-
== Dilution decreasing GFP concentration ==
 
-
At the start of our measurements, the GFP protein has reached a steady state high concentration.
 
-
The specific GFP will decrease during growth because of dilution, and this needs to be accounted for when calculating the RBS strength.
 
-
[[Image:TUD2010_Gfpod_all.png]]
+
=== Data normalization method ===
 +
To go from the raw measurements to the RBS strengths, we ran the following steps using a matlab script:
-
== Protein production model ==
+
[[Image:TUDelft2010_Rbs-control-gfp.png|right|thumb|300px]]
-
As said, the protein production model needs to take dilution into account. Since the used GFP is a very stable protein, the degradation term is negligible compared to dilution.
+
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
-
[[Image:RBS_Expression_model.PNG]]
+
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:
 +
[[Image:TUDelft_2010_PPM_Autofluorescence.png]]
 +
* 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]].
-
In above formula:
+
<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>
-
* Y is the GFP concentration
+
-
* Beta is the production rate
+
-
* Alpha is the dilution+degradation rate
+
-
 
+
-
Converting this equation to an explicit form results in:
+
-
 
+
-
 
+
-
 
+
-
Go to [[Team:TU_Delft/Project/rbs-characterization/results|results]] for measured RBS strengths
+
-
 
+
-
== Source code and data ==
+
-
https://static.igem.org/mediawiki/2010/8/88/TUDelft_2010_RBS_characterization.zip
+
-
 
+
-
== References ==
+
-
;Kelly 2009
+
-
: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
+

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

Retrieved from "http://2010.igem.org/Team:TU_Delft/Project/rbs-characterization/characterization"