Team:TU Delft/Project/rbs-characterization

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(Ribosome Binding Site Characterization)
 
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{{Team:TU_Delft/frame_check}}
{{Team:TU_Delft/frame_check}}
==Ribosome Binding Site Characterization==
==Ribosome Binding Site Characterization==
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Part of our project is to measure the effect of various ribosome binding site (RBS) sequences. A RBS sequence is a specific mRNA sequence that folds in such a way that it attracts the ribosome. This ribosome binds to the mRNA molecule and starts translation of the mRNA into protein. Accurate information on how well this binding occurs (The binding strength) can be very useful when designing new biological systems.
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[[Image:TUDelft_RBS.png|300px|right]]
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A ribosomal binding site sequence is a specific mRNA sequence that folds in such a way that it attracts the ribosome. This ribosome binds to the mRNA molecule and starts translation of the mRNA into protein.
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We took five different RBS sequences from the [http://partsregistry.org/Ribosome_Binding_Sites/Prokaryotic/Constitutive/Anderson Anderson RBS family] (J61100, J61101, J61107, J61117, J61127). The standard RBS [http://partsregistry.org/Part:BBa_B0032 B0032] was used as a reference for our characterization.  
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As with many synthetically implemented pathways, the rate of conversion through the pathway is highly influenced by the expression levels of the proteins involved. While the way in which these protein levels have their influence on the rates is not yet fully understood, the simple bottle-neck explanation has generally been accepted in the community.
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All these RBS sequences were placed in front of the standard [http://partsregistry.org/Part:BBa_E0040 GFP coding sequence], so expression could be measured.
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Consider the following pathway:  
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The Biobricks generated in order to perform the experiments were: [http://partsregistry.org/Part:BBa_K398500 K398500], [http://partsregistry.org/Part:BBa_K398501 K398501], [http://partsregistry.org/Part:BBa_K398502 K398502], [http://partsregistry.org/Part:BBa_K398503 K398503], [http://partsregistry.org/Part:BBa_K398504 K398504]. The general map of the construction is shown below, where the RBS is displayed in fucsia.
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#alkane → alkanol ; catalyzed by LadA
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#alkanol → alkanal ; catalyzed by ADH
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#alkanal → alkanoic acid ; catalyzed by ALDH
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[[Image:RBS1.jpg|left|650px]]
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If, for example, the protein expression level of ALDH is lower than that of ADH, a build-up of alkanal occurs, which will be toxic to the cell. Thus, in implementing our alkane degradation pathway we aimed to increase the protein levels in accordance with the conversion steps.
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A perfect way to implement this variation in the alkane degradation pathway is by using ribosome binding sites with varying strengths. This led to the characterization of the ribosome initiation strengths of five Anderson RBS family members.
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{| style="color:black; background-color:white;" cellpadding="5" cellspacing="0" border="1"  
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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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|'''Feature'''
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|'''Function'''
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|[http://partsregistry.org/Part:BBa_B0032 AmpR]
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|Ampicillin resistance
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|[http://partsregistry.org/Part:BBa_B0015 B0015]
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|Transcriptional (double) terminator
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|-
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|[http://partsregistry.org/Part:BBa_B0062 B0062]
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|Transcriptional terminator
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|-
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|[http://partsregistry.org/Part:BBa_E0040 E0040]
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|GFP
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|-
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|[http://partsregistry.org/Part:BBa_G00000 G00000]
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|Standard prefix
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|-
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|[http://partsregistry.org/Part:BBa_G00001 G00001]
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|Standard suffix
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|-
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|[http://partsregistry.org/Part:BBa_G00100 G00100]
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|VF2 primer binding site
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|-
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|[http://partsregistry.org/Part:BBa_G00101 G00101]
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|VR primer binding site
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|-
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|[http://partsregistry.org/Part:BBa_J61100 J61100]
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|RBS Anderson family
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|-
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|[http://partsregistry.org/Part:BBa_J23100 J23100]
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|Promoter
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|}
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<html><div style='clear:both'>
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</html>
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* [[Team:TU_Delft/Project/rbs-characterization/parts|Parts]]
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* [[Team:TU_Delft/Project/rbs-characterization/characterization|Characterization]]
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* [[Team:TU_Delft/Project/rbs-characterization/results|Results and Conclusions]]
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Latest revision as of 19:59, 24 October 2010

Ribosome Binding Site Characterization

TUDelft RBS.png

A ribosomal binding site sequence is a specific mRNA sequence that folds in such a way that it attracts the ribosome. This ribosome binds to the mRNA molecule and starts translation of the mRNA into protein.

As with many synthetically implemented pathways, the rate of conversion through the pathway is highly influenced by the expression levels of the proteins involved. While the way in which these protein levels have their influence on the rates is not yet fully understood, the simple bottle-neck explanation has generally been accepted in the community.

Consider the following pathway:

  1. alkane → alkanol ; catalyzed by LadA
  2. alkanol → alkanal ; catalyzed by ADH
  3. alkanal → alkanoic acid ; catalyzed by ALDH

If, for example, the protein expression level of ALDH is lower than that of ADH, a build-up of alkanal occurs, which will be toxic to the cell. Thus, in implementing our alkane degradation pathway we aimed to increase the protein levels in accordance with the conversion steps.

A perfect way to implement this variation in the alkane degradation pathway is by using ribosome binding sites with varying strengths. This led to the characterization of the ribosome initiation strengths of five Anderson RBS family members.

CharacterizationResultsParts