Instructions

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 rationally control the production rate of any protein in bacteria from 0.1 to 100,000+ on a proportional scale.

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 inside the cell 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 and one of the last steps 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 its messenger RNA transcript. The coding sequence of the mRNA transcript controls which protein is produced while the non-coding sequence controls how much protein is produced per second. Specifically, there is a relatively short non-coding nucleotide 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 the start codons. 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

·        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 ATG or GTG and any combination of ATCGU nucleotides is acceptable. Thymines (T) are automatically converted to uracils (U).

·        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.

·        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.

·        Step 4: Enter your email address and click on the "RBS Design" button. A link to the results will be emailed to you in about 5 to 15 minutes.

The results will contain the pre-sequence, the ribosome binding site sequence, and the protein coding sequence with the predicted translation initiation rate. On average, the actual translation initiation rate will be within 2.3-fold of the predicted rate. Importantly, this prediction is specific to the protein coding sequence. If you change the protein coding sequence, a new ribosome binding site should be designed.

Forward Engineering Mode with RBS 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.

·        Step 1: Add a sequence constraint in the “RBS Constraints” box. Enter any combination of ATCG nucleotides to keep a nucleotide constant. Enter an ‘N’ nucleotide 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.

·        Step 2: Specify the protein coding sequence, pre-sequence, and target translation initiation rate in the forward engineering mode. Verify that the length of the pre-sequence and RBS sequence constraint adds up to at least 50 nucleotides.

·        Step 3: Enter your email address and click on the “RBS Design” button. A link to your results will be emailed to you in about 5 to 60 minutes. The

Reverse Engineering Mode

·        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).

·        Step 2: Enter your email address and click on the "RBS Predict" button. A link to the results will be emailed to you in about 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 a green box ("OK"). For various reasons, the accuracy of a prediction made in the reverse engineering mode may be less than a prediction made in the forward engineering mode. legend contains a list of reasons.