- The Ribosome Binding Site (RBS) Calculator is a design method for predicting and controlling translation initiation and protein expression in bacteria.
- In its reverse engineering mode, RBS calculator predicts the rate of translation initiation for every start codon in an mRNA transcript.
- In its forward engineering mode, RBS calculator optimizes a synthetic RBS sequence to achieve a targeted translation initiation rate.
- Using the RBS Calculator, a protein coding sequence’s (CDS) translation rate may be rationally controlled across a 100,000þ fold range.
- This method employs a thermodynamic model of bacterial translation initiation to calculate the Gibbs free energy of ribosome binding.
Applications of the RBS calculator:
1. Manipulating the protein expression level - The RBS Calculator allows production of protein of interest at a selected expression level in bacteria (e.g., E. coli BL21 or C43) by balancing the translation rate with the transcription and folding rates.
2. Optimizing synthetic metabolic pathways - Bacteria employ synthetic metabolic pathways to manufacture high-value chemical products from sugars or plant biomass. The RBS Calculator efficiently identifies the optimal enzyme expression levels of a metabolic pathway, eliminating any bottlenecks in the pathway, and maximizing its productivity.
3. Designing and connecting genetic circuits - The RBS Calculator allows a rational tune the expression levels of the proteins in a genetic circuit, either during the initial design phase or when connecting two genetic circuits together
4. Evolutionary robustness of RBSs - During evolutionary mutation and selection, protein expression level changes can result in physiological changes that increase an organism’s fitness. The RBS Calculator’s reverse engineering mode performs a sensitivity analysis on a RBS sequence, identifying which nucleotide mutations will most affect its translation initiation rate and protein expression level. Thus, probable paths during evolution can be identified.
5. Predicting translation initiation rates across a genome - The RBS Calculator’s reverse engineering mode can predict the translation initiation rate of every start codon in a bacterial genome. These predictions enable you to find the correct start codons of open reading frames, estimate the expression levels of proteins within an operon, and identify internal start codons that exhibit significant translation and produce variant proteins. .