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<center><h1> Instructions</h1></center> | <center><h1> Instructions</h1></center> | ||
− | <h3>Create Your Account</h3> | + | <p><h3>Create Your Account</h3></p> |
− | <img src="https://static.igem.org/mediawiki/2017/d/de/CUHK_register.png" width="20%" height="auto" style="float: left"> | + | <img src="https://static.igem.org/mediawiki/2017/d/de/CUHK_register.png" width="20%" height="auto" style="margin: 0px 10px; float: left"> |
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
− | + | <br><br> | |
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. | 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. | ||
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− | < | + | <p><h3>Design your toehold switch</h3></p> |
+ | <p><h4><u>Input target RNA sequence</u></h4></p> | ||
<p style="font-family: roboto;font-size:115%;"> | <p style="font-family: roboto;font-size:115%;"> | ||
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User can input target RNA in plain text or FASTA file with numbers, space, newline, uppercase, lowercase, T or U. | 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. | 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. | 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 | + | 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: |
− | < | + | <ul> |
− | + | <li>Have no C or G at the neck of hairpin </li> | |
− | < | + | <li>Have in frame stop codon after AUG </li> |
− | + | <li>Have more than four consecutive A, G, C or T </li> | |
− | < | + | </ul> |
− | + | <br><p style="font-family: roboto;font-size:115%;"> | |
− | <br> | + | |
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. | 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. | ||
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− | <h4>Input parameters:</h4> | + | <p><h4><u>Input parameters:</u></h4></p> |
− | + | <ul> | |
− | < | + | <li><b>Choose your cloning method (optional) </b> |
− | User can choose to add RE site or use our toehold switch cloning tool. | + | <p style="font-family: roboto;font-size:100%;"> |
− | < | + | User can choose to add RE site or use our toehold switch cloning tool. </i></li> |
− | + | <li><b>Choose Promoter (optional) </b> | |
− | < | + | <p style="font-family: roboto;font-size:100%;"> |
− | User can input a custom promoter or select a promoter from iGEM registry. | + | User can input a custom promoter or select a promoter from iGEM registry.</i> </li> |
− | < | + | <li><b>Input linker sequence </b> |
− | + | <p style="font-family: roboto;font-size:100%;"> | |
− | < | + | The linker is used to separate the coding sequence in the toehold switch and the reporter to prevent interference of protein folding. A default flexible linker (AACCUGGCGGCAGCGCAAAAG) is provided. </i></li> |
− | The linker is used to separate the coding sequence in the toehold switch and the reporter to prevent interference of protein folding. A default flexible linker (AACCUGGCGGCAGCGCAAAAG) is provided. | + | <li><b>Choose ribosomal binding site </b> |
− | < | + | <p style="font-family: roboto;font-size:100%;"> |
− | + | User can input a custom RBS or select a RBS from iGEM registry.</i> </li> | |
− | < | + | <li> <b>Input trigger length</b> |
− | User can input a custom RBS or select a RBS from iGEM registry. | + | <p style="font-family: roboto;font-size:100%;"> |
− | < | + | The downstream 120nt (or user specified length) from the start site will be copied to be trigger sequence. </i></li> |
− | + | <li><b>Check RFC</b> <br> | |
− | < | + | <i>RFC standard check are specifically added for iGEMers. The program can help you to eliminate switches with illegal sites.</i> |
− | The downstream 120nt (or user specified length) from the start site will be copied to be trigger sequence. | + | </li> |
− | < | + | </ul> |
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− | RFC standard check are specifically added for iGEMers. The program can help you to eliminate switches with illegal sites. | + | |
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+ | <p><h4><u>Output option </u></h4></p> | ||
+ | <ul> | ||
+ | <li><b>Counting rare codon</b> | ||
+ | <p style="font-family: roboto;font-size:100%;"> | ||
+ | User can choose to count the occurrence of rare codon at the start of the CDS. Rare codon at the beginning of CDS may significantly hinder translation. </i></li> | ||
+ | </i></li> | ||
+ | <li><b>Minimal free energy (MFE) and MFE structure calculation</b> | ||
+ | <p style="font-family: roboto;font-size:100%;"> | ||
User can choose to output the MFE and MFE structure of this switch, switch dimer, window-switch dimer and RBS-linker. We used embedded function of <a href="https://www.tbi.univie.ac.at/RNA/">“Vienna RNA”</a> (1) to do the calculation. | User can choose to output the MFE and MFE structure of this switch, switch dimer, window-switch dimer and RBS-linker. We used embedded function of <a href="https://www.tbi.univie.ac.at/RNA/">“Vienna RNA”</a> (1) to do the calculation. | ||
− | < | + | </i></li> |
− | + | <li><b>Base pair condition</b> | |
− | < | + | <p style="font-family: roboto;font-size:100%;"> |
The base pair condition of the toehold domain of switch sequence and the RBS domain in trigger-switch dimer will be counted, and switches with excessive domain pair will be discarded. A low number of base pairing at the toehold domain and RBS is preferred. | The base pair condition of the toehold domain of switch sequence and the RBS domain in trigger-switch dimer will be counted, and switches with excessive domain pair will be discarded. A low number of base pairing at the toehold domain and RBS is preferred. | ||
− | < | + | </i></li> |
− | + | <li> <b>BLASTn</b> | |
− | < | + | <p style="font-family: roboto;font-size:100%;"> |
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. | 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. | ||
− | < | + | </i></li> |
+ | </ul> | ||
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+ | <p><h3>Output</h3></p> | ||
+ | <p><h4><u> | ||
+ | Excel output</h4></u></p> | ||
− | + | <p style="font-family: roboto;font-size:115%;"> | |
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The output result will be represented in a excel file. For example: </p> | The output result will be represented in a excel file. For example: </p> | ||
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− | + | <p><h4><u> Email output </h4></u></p> | |
− | < | + | <p style="font-family: roboto;font-size:115%;"> |
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. | 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. | Result files can also be retrieved in user account. The upper limit of fiel number is fifty, including excel files and BLASTn files. |
Revision as of 09:14, 31 October 2017