What is the RBS Calculator?

The Ribosome Binding Site (RBS) Calculator is an engineering design method that predicts the translation initiation rate of a protein coding sequence in bacteria. You can use the RBS Calculator to generate synthetic ribosome binding site sequences and to rationally control the production rate of any protein in bacteria from 0.1 to 100,000+ on a proportional scale.

You may use the RBS Calculator here.

Introduction

Proteins are the workhorse of cellular organisms. They sense the environment outside a cell, regulate the expression of genes within the cell's genome, and catalyze the chemical reactions that control the cell's metabolism. Different proteins will have different activities and the strength of those activities will depend on the amount of protein present. Proteins are produced in a multi-step process called gene expression, where DNA is transcribed into messenger RNA (mRNA), which is in turn translated into protein. This last step of gene expression is called translation. In translation, the protein-making machinery (called the ribosome) is instructed to produce a specific protein according to the nucleotide sequence of a mRNA transcript. This nucleotide sequence is called the protein coding sequence. The protein coding sequence controls which protein is produced while the non-coding portion of the mRNA sequence controls how much protein is produced per second. Specifically, there is a relatively short sequence in front of each protein coding sequence that controls how many times the translation process is initiated per second. This non-coding sequence controls the translation initiation rate and is called the ribosome binding site (RBS). By modifying this sequence, we can change the protein's translation initiation rate, proportionally alter its production rate, and control its activity inside the cell.

How to Use the RBS Calculator

The RBS Calculator can be used in either Reverse Engineering or Forward Engineering modes. In the Reverse Engineering mode, you enter the nucleotide sequence of an mRNA transcript and the RBS Calculator predicts the translation initiation rate from each of its start codons, either AUG, GUG, or UUG. In the Forward Engineering mode, you enter a protein coding sequence and a target translation initiation rate. The RBS calculator then generates a synthetic ribosome binding site sequence that will initiate your protein coding sequence at the selected translation initiation rate.

Forward Engineering Mode

  1. Step 1: Copy and paste the first 50 nucleotides of a protein coding sequence into the Protein Coding Sequence box. The sequence must start with either AUG or GUG and any combination of ATCGU nucleotides is acceptable. Thymines (T) are automatically converted to uracils (U).
  2. Step 2: Select the target translation initiation rate, which is gauged on a proportional scale from 0.1 to 100,000+. There is no lower or upper limit to the scale and translation rates below 0.1 or above 100,000 are feasible, depending on the protein coding sequence.
  3. Step 3: Input the pre-sequence, which is any sequence that appears prior to the ribosome binding site, such as a restriction site. The sequence must be a part of the mRNA transcript.
  4. Step 4: Click on the "Submit Job" button. The result will be stored in your "My Results" list and will be completed within 1 to 15 minutes, depending on the selected translation initiation rate.

The results will contain the pre-sequence, the synthetic ribosome binding site sequence, and the protein coding sequence. The translation initiation rate of the synthetic ribosome binding site / start codon and of any other start codons in the mRNA sequence will be displayed. Due to RNA-RNA interactions between the ribosome binding site and protein coding sequence, if you change the protein coding sequence, you should also design a new synthetic ribosome binding site.

Forward Engineering Mode with Constraints

Sometimes, it is necessary to insert a specific sequence within or nearby the ribosome binding site (e.g. a restriction site or a primer binding site). You can design a synthetic ribosome binding site and include such sequences by adding a sequence constraint.

  1. Step 1: Add a sequence constraint in the RBS Constraints box. Enter any combination of ATCGU nucleotides to keep a nucleotide constant. Enter an N to allow a nucleotide to change. For example, the sequence constraint TCTAGANNNNNNNNNNNNNNN will generate a 21 nucleotide synthetic RBS sequence that begins with the XbaI restriction site.
  2. Step 2: Similar to the forward engineering mode, specify the protein coding sequence, pre-sequence, and target translation initiation rate. Verify that the length of the pre-sequence and RBS sequence constraint adds up to at least 50 nucleotides.
  3. Step 3: Click on the "Submit Job" button. The result will be stored in your "My Results" list and will be completed within 1 to 45 minutes, depending on the selected translation initiation rate and RBS Constraints. A solution may not exist if a a short or highly constrained RBS sequence is entered and/or a very high translation initiation rate is selected.

Reverse Engineering

  1. Step 1: Copy and paste the sequence of an mRNA transcript into the mRNA Sequence box. The sequence should contain at least 50 nucleotides before and after the start codon of your protein(s) of interest. If a start codon is located near the beginning (5' end) of the mRNA transcript, only include the nucleotides in the mRNA transcript. Any combination of ATCGU nucleotides is acceptable. Thymines (T) are automatically converted to uracils (U).
  2. Step 2: Click on the "Submit Job" button. The results will be stored in your "My Results" list and will be completed in less than 5 minutes.

The results will contain a list of start codons in the mRNA transcript and their predicted translation initiation rates on a relative scale from <1 to 100,000+. Each start codon will produce a different protein. The accuracy of the prediction is denoted by a colored box and abbreviation. An accurate prediction is denoted by OK. For various reasons, a prediction may be less accurate than normal. For example, if a mRNA may not fold quickly to its equilibrium state, then a "Not At Equilibrium" (NEQ) warning is issued. If two start codons are too close together, then a "Overlapping Start Codons" (OLS) warning is issued. Finally, if the protein coding sequence is too short to make an accurate prediction, then a "Short CDS" (CDS) warning is issued. A CDS warning may be corrected by inputting a longer protein coding sequence.