Frequently Asked
Questions
Didn’t find your question? Contact Howard Salis.
Q: How does the RBS Calculator work?
The RBS Calculator combines a thermodynamic model of bacterial translation initiation with an optimization algorithm for sequence design. The thermodynamic model accepts an arbitrary mRNA sequence and uses a free energy model to calculate the total change in Gibbs free energy when the 30S complex of the ribosome binds to a ribosome binding site. The free energy model includes five terms that quantify the strengths of the important molecular interactions involved in translation initiation. The predicted Gibbs free energy change is then related to the relative translation initiation rate and reported on a proportional scale from 0.1 to 100 000+. For additional information, please see the article describing the method1.
Q: What does the proportional scale mean and why not use absolute units?
All RBS sequences are compared using the same proportional scale, which quantifies the translation initiation rate of an RBS sequence, but not the remaining steps of gene expression. Using the scale, the translation initiation rates between any two RBS sequences can be compared. Synthetic RBS sequences can also be rationally designed to increase or decrease the translation initiation rate of any protein coding sequence by a desired factor. However, without a predictive model of the remaining steps of gene expression, it is not currently possible to predict the absolute rate of protein production.
Q: Why do I need to input the first 50 nucleotides of the protein coding sequence to generate a synthetic ribosome binding site?
The translation initiation rate depends not only on the ribosome binding site, but also on the protein coding sequence. In fact, reusing the same ribosome binding site sequence with different protein coding sequences can result in over a 500-fold change in translation initiation rate1. These effects are caused by the formation of RNA secondary structures that prevent the ribosome from binding, altering the translation initiation rate. The types and strengths of these secondary structures depend both on the ribosome binding site and the protein coding sequence. No matter which ribosome binding site sequence is chosen there is always a protein coding sequence that will form a particularly strong secondary structure. Therefore, to obtain an accurate prediction, the protein coding sequence must also be specified.
Q: How accurate is the RBS Calculator?
On average, the predictions of the RBS Calculator are accurate to within a factor of 2.3, equivalent to an error of 1.82 kcal/mol in the thermodynamic model. There is a 47% chance that a synthetic ribosome binding site will be accurate to within 2-fold of its predicted translation initiation rate. This probability can be increased to 72%, 85%, or 92% by generating two, three, or four additional synthetic ribosome binding site sequences with the same target rate.
Q: In which organisms will the RBS Calculator work well?
The thermodynamic model of bacterial translation initiation was developed and tested in Escherichia coli, a gram-negative bacterium in the Enterobacteriaceae family of gamma-proteobacteria. The thermodynamic model has also been utilized in Klebsiella M5AL and Salmonella enterica serovar Typhi, which are both gram-negative bacterium in gamma-proteobacteria. While additional measurements in other species is required, most gram-negative bacteria have strong similarities in their translation machinery, suggesting that the RBS Calculator will be roughly accurate within these related bacteria. However, the translation machinery in gram-positive bacteria is known to differ in some characteristics; additional characterization will be necessary to accurately predict the translation rate of RBS sequences in gram-positive bacteria.
References
1. Salis, H., Mirsky, E.A. & Voigt, C.A. Automated Design of Synthetic Ribosome Binding Sites to Precisely Control Protein Expression. Nat Biotechnol (accepted).