"

From 2010.igem.org

One of the major problems that past teams were faced was about not having fine control over protein expression. As RBS sequence play significant role on rate of protein expression, by designing a particular RBS for a specific protein we can literally over come this problem.

Ribosome Binding Site

The Ribosome Binding Site (RBS) is a sequence on the messenger RNA to which the ribosome can bind and initiate protein translation. In bacteria, translation initiation requires an RBS sequence and a start codon. Ribosome binding sites can control the translation initiation rate and thereby protein expression1. Regulating the level of protein expression is required to connect genetic circuit1 and control flux through a metabolic pathway2. The RBS affects the translation rate of an open reading frame (ORF) in two main ways3. Firstly, the rate at which ribosomes are attached to the mRNA and initiate translation is dependent on the RBS sequence. Secondly, the RBS influences the level of protein synthesis by modifying mRNA stability. The stability of the mRNA has impact on the steady state level of mRNA; a stable mRNA will have a higher steady state level than an unstable mRNA that is being produced as an identical rate. Since the primary sequence and the secondary structure of an RBS (for example, the RBS could introduce a RNase site) can affect the stability of the mRNA, the RBS can affect the amount of mRNA therefore, the level of protein expression.

Translation in Bacteria involves four steps: initiation, elongation, termination and ribosome turnover4. Translation initiation is the rate-limiting step. Its rate is determined by molecular interactions, such as hybridization of the 16s RNA to the RBS sequence, the binding site of tRNAfMET to the start codon, the distance between the 16s rRNA binding site and the start codon (spacing) and the presence of RNA secondary structures that occlude either the 16s rRNA binding site or the standby site1. A thermodynamic model takes into account the strength of molecular interaction between an mRNA and the 30S ribosome complex to predict the translation initiation rate1. The model is based on two separated reversible transition states. The initial state is the folded mRNA and the free 30S complex and the final state is the assembled 30S complex on an mRNA. The difference in Gibbs free energy between these two states (ΔGtot) relies on the mRNA sequence surrounding a start codon (AUG or GUG). ΔGtot is more negative when there is an attractive interaction between ribosome and mRNA and ΔGtot is more positive when there is exclusive secondary structure1.

How we can measure and regulate protein expression

The RBS Calculator is a piece of software programmed to design synthetic ribosome binding sites, facilitating a rational control of protein expression. The software has two parts: forward engineering and reverse engineering. Forward engineering incorporates a thermodynamic model into an optimizing algorithm for designing a ribosome binding site sequence which is predicted to drive protein translation at a particular rate. Reveres engineering predicts the relative translation rate of an existing RBS sequence with regard to upstream protein coding sequence.

[http://voigtlab.ucsf.edu/software/ RBS Calculator]

Reference

1. Voigt, C., Mirsky, E., Salis, H. Automated desing of sysnthetic ribosome binding sites to control protein expression. Nature Biotechnology ,946-950 (2009)

2. Dueber, J.E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nature. Biotechnolgy. 27, 753–759 (2009)

3. Bernstein, JA., Khodursky, AB., Lin, PH., Lin-Chao, S., Cohsen, SN., Global analysis of mRNA decay and abundance in Escherichia coli at single-gene resolution using two-color fluorescent DNA microarrays.