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

From 2010.igem.org

(Difference between revisions)
(Growth curve fitting)
(Protein production model)
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In above formula:
In above formula:
* Y is the GFP concentration
* Y is the GFP concentration
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* Beta is the production rate
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* Beta is the production rate (The constant that we are interested in)
* Alpha is the dilution+degradation rate
* Alpha is the dilution+degradation rate
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The 2 unknown parameters (beta and Y1) where curve-fitted against the GFP/OD data, using the matlab lsqcurvefit command.
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.
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Go to [[Team:TU_Delft/Project/rbs-characterization/results|results]] for measured RBS strengths
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== Source code and data ==
== Source code and data ==

Revision as of 14:58, 7 October 2010

Contents

Characterization

RBS strength

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:

  • The rapid growth of the bacteria will dilute the protein considerably
  • Proteins degrade over time.

Growth curve fitting

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.

Tud2010 RBS OD.png

The exponential part of this graph is curve-fitted to an exponential growth model:

TUD2010 RBS Growth model.PNG

Which results in a growth rate ('mu') that can be used to add dilution to further calculation.

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.

TUD2010 Gfpod all.png

Protein production model

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.

RBS Expression model.PNG

In above formula:

  • Y is the GFP concentration
  • Beta is the production rate (The constant that we are interested in)
  • Alpha is the dilution+degradation rate

Converting this equation to an explicit form results in:

TU2010 Gfp explicit.PNG

The 2 unknown parameters (beta and Y1) where curve-fitted against the GFP/OD data, using the matlab lsqcurvefit command. The beta constant for each of the 72 experiments were then to calculate RBS strengths, listed in the results page.

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). "Measuring the activity of BioBrick promoters using an in vivo reference standard". Journal of Biological Engineering 3: 4