Software overview

To reduce manpower, we wrote a program to automatically generate toehold switch sequences from target RNA in put. Then we developed it to a website for the convenience of other iGEMers who are interested in toehold switch application. Though this website can execute many functions to reduce repetitive works for user, careful examination of thermodynamic parameters and RNA secondary structures is still encouraged to get a promising switch sequence (For more information, please visit RNA thermodynamics modelling page). Suboptimal structure analysis is particularly recommended. This function is not included in the main program because it’s too time consuming. However, iGEMers could get access to a professional website with suboptimal structure calculation function through our link under “Model” tab. To help iGEMers get started, we would like to do a brief introduction on our website here.

Instructions

Create Your Account

An account is required for every user to execute the main program, save the result and retrieve result later. A total of fifty results could be saved in one account.

Input target RNA sequence

On the “Design toehold switch” page, user can input target RNA in plain text or FASTA file with numbers, space, newline, uppercase, lowercase, T or U. Target RNA sequences will be transformed to uppercased DNA sequence. All downstream process will use this format. Sequences less than 30 bp will be rejected. After procession of input, a page with all user inputs will prompt for user to check and confirm. Rejected sequences will also be shown. The program will set the first nucleotide of each target RNA as the start point, generate possible switch sequences for each 30, 31, 32, 33, 34 or 35 nucleotides (nt), which is called a window sequence. Since the whole switch sequence could be predicted by the initial window sequence, window sequence with following features will be rejected to ensure hairpin stability,:
• Have no C or G at the neck of hairpin
• Have in frame stop codon after AUG
• Have more than four consecutive A, G, C or T
Start point will move to right for one nucleotide if the previous window sequence is rejected. Switch design process will continue if the window sequence passed the first examination.

Ribosomal binding site

Then “AUA” or “UAU”, ribosomal binding site (RBS), “UAU” or “AUA” and the second half of the hairpin will be added after window sequences.

Rare codon count and RFC standard check

MFE calculation

MFE and MFE structure of this switch, switch dimer, window-switch dimer and RBS-linker will be calculated according to user input. The base pair condition of the toehold domain of switch sequence will be counted, and switches with excessive domain pair will be discarded.

Trigger length

The downstream 120 nt (or user specified length) from the start site will be copied to be trigger sequence. Trigger sequence will co-express with corresponding switch under the control of T7 promoter to validate the functionality of the switch. Then MFE and MFE structure of trigger, trigger dimer, trigger-switch dimer will be calculated as user input.

Plasmid construction

For plasmid construction, three options are provided. The first option is to use our standard backbone, which contains T7 promoter, EcoR31 recognition site and antibiotics resistance gene. A plasmid for specific switch and trigger pair can be produced by simply digesting the switch standard backbone and trigger standard backbone and ligating them with switch and trigger separately. The second option is simple output switch and trigger sequence. The third option is generate overlapping PCR primers for user.

BLASTn

After all target RNA sequences are processed, BLASTn will be done for every legal window sequences if user ticked this option. BLASTn result will be saved in txt file with switch number as file name.

Output excel file

The output result will be represented in a excel file. Below list out the definition of terms used in the output excel.

Email address

User will receive result files by email. If the input is only one target RNA sequence without BLASTn function, the email with contain an excel file named by user specified name. If the input is multiple sequences or BLASTn is required, the email will contain a folder named by user specified name. Within the folder, both excel files for each target RNAs and BLASTn files will be named in digit numbers. Result files can also be retrieved in user account. The upper limit of fiel number is fifty, including excel files and BLASTn files.