Team:TU Delft/Project/rbs-characterization


Revision as of 17:32, 22 October 2010 by Mvoges (Talk | contribs)

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.

For example, consider the pathway A → B, where the protein expression level of B is lower than that of A, due to the slower conversion by B, a build-up of intermediates occurs, which could be detrimental to the cell (depending on the toxicity of the intermediate). Thus, in implementing our alkane degradation pathway we aimed to increase the protein levels in accordance with the conversion steps.

In order to achieve this the ribosome initiation strengths of five Anderson RBS family members were characterized. By varying only the RBS sequence in a protein generator construct the dependence of the expression level of the encoded protein on the RBS sequence could be determined by simple fluorescence and biomass curve analysis.

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