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

From 2010.igem.org

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==Characterization==
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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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</div></html>
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[[Image:26_07_2010_Rbs.png|center]]
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'''Note:''' Fluorescence (y-axis) is reported as Arbitrary Fluorescence Units.
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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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== Plots of measurements ==
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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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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1" align="center"
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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. The graph below shows 72 growth curves. 12 seperate wells were used for every RBS sequence.
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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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|[http://partsregistry.org/Part:BBa_J61107 J61107]
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|0.065454
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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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[[Image:Tud2010_RBS_OD.png]]
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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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== Dilution decreasing GFP concentration ==
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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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[[Image:TUD2010_Gfpod_all.png]]
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=== Source data ===
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== Protein production model ==
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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.
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Source code and data used for characterization:
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[[Image:RBS_Expression_model.PNG]]
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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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In above formula:
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* Y is the GFP concentration
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* Beta is the production rate
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* Alpha is the dilution+degradation rate
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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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Converting this equation to an explicit form results in:
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Go to [[Team:TU_Delft/Project/rbs-characterization/results|results]] for measured RBS strengths
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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

Revision as of 14:12, 7 October 2010

Contents

Characterization

Plots of measurements

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.

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.

Tud2010 RBS OD.png

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. Since the used GFP is a very stable protein, 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
  • Alpha is the dilution+degradation rate

Converting this equation to an explicit form results in:


Go to results for measured RBS strengths

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