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| − | <h1 class="sectionTitle-L firstTitle">Protein Optimization & Gibson Assembly Primer design</h1> | + | <h1 class="sectionTitle-L firstTitle">pABA Elementary Flux Mode Analysis</h1> |
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| − | <div class="col-sm-12 pagetext">The purpose of this script is to expedite the process of protein DNA sequence optimization and Gibson Primer design for Gibson assembly reactions. The files for the script can be found either <a href="https://github.com/gsun1729/Prot-Optimization-Gibson-Assembly">here on github</a> or be downloaded individually from the IGEM database below. <br><br> | + | <div class="col-sm-12 pagetext">We wanted to use elementary flux mode analysis to identify targets for gene knockout/gene overexpression. Elementary flux mode analysis allows us to calculate the elementary flux modes, which are analogous to the 'metabolic freedom' of the metabolic model. Using the EFMs, we are able to understand all of the "potential capabilities" of the organism. We then examine the metabolic network for different things we want to have done, and things we want to eliminate in order to increase pABA production. Using this, we select targets for knockout/overexpression. |
| − | Note because IGEM does not support .py file extensions, you will need to remove .txt from the extensions of all of the downloaded files from the links below and append ".py" to the end of (1) seq_analyzer, (2) seq_tools, (3) input_tools, and (4) format_tools (i.e. "seq_tools.txt" becomes "seq_tools.py.") No further formatting is needed for the github link download.<br>
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| − | <ul>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/1/1f/T--Stanford-Brown--POGAS_seq_analyzer.txt">seq_analyzer</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/b/b7/T--Stanford-Brown--POGAS_seq_tools.txt">seq_tools</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/e/e5/T--Stanford-Brown--POGAS_input_tools.txt">input_tools</a>
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| − | <li><a href="https://2016.igem.org/File:T--Stanford-Brown--POGAS_format_tools.txt">format_tools</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/d/d1/T--Stanford-Brown--POGAS_NT_Lib.txt">NT_Lib</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/6/60/T--Stanford-Brown--POGAS_codonlib.txt">Codon_Lib</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/9/90/T--Stanford-Brown--POGAS_restriction_enzymes.txt">restriction_enzymes</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/d/d5/T--Stanford-Brown--POGAS_ecoli.txt">ecoli</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/4/41/T--Stanford-Brown--POGAS_Ecolik12.txt">ecoliK12</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/f/f5/T--Stanford-Brown--POGAS_yeast.txt">yeast</a>
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| − | </li>
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| − | </ul>
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| − | </div> <!--END col-sm-12-->
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| − | <div class="col-sm-12 pagetext">
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| − | Sample input files are also included in the repository and are listed below
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| − | <ul>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/1/1f/T--Stanford-Brown--POGAS_seq_analyzer.txt">seq_analyzer</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/b/b7/T--Stanford-Brown--POGAS_seq_tools.txt">seq_tools</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/e/e5/T--Stanford-Brown--POGAS_input_tools.txt">input_tools</a>
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| − | <li><a href="https://2016.igem.org/File:T--Stanford-Brown--POGAS_format_tools.txt">format_tools</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/d/d1/T--Stanford-Brown--POGAS_NT_Lib.txt">NT_Lib</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/6/60/T--Stanford-Brown--POGAS_codonlib.txt">Codon_Lib</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/9/90/T--Stanford-Brown--POGAS_restriction_enzymes.txt">restriction_enzymes</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/d/d5/T--Stanford-Brown--POGAS_ecoli.txt">ecoli</a>
| + | Product_max_without_RX = 49.6064 |
| − | <li><a href="https://static.igem.org/mediawiki/2016/4/41/T--Stanford-Brown--POGAS_Ecolik12.txt">ecoliK12</a>
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| − | <li><a href="https://static.igem.org/mediawiki/2016/f/f5/T--Stanford-Brown--POGAS_yeast.txt">yeast</a>
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| − | </li>
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| − | </ul>
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| − | </div> <!--END col-sm-12-->
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| − | <h2 class="subHead">Input File Formatting</h2>
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| − | <div class="row">
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| − | <div class="col-sm-12 pagetext">In order to run the program, the user has to provide a text file containing the sequences in need of optimization and/or primer design. Each sequence should be included on a separate line; any blank newline entries and spaces will result in a processing error. The sequences should also contain no other characters other than that representing nucleotides (A,T,G,C) or amino acids (G,A,L,M,F,W,K,Q,E,S,P,V,I,C,Y,H,R,N,D,T,*).
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| − | <br><br>
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| − | For protein optimization, sequences in the input file are not limited to only amino acid or nucleotide sequences--the user can input either and the program will recognize the sequence type and process it accordingly. This is limited however in cases where the only amino acids in the sequence are alanine, threonine, cysteine, and glycine, since the single letter code for each amino acid is also found in the single letter nucleotide representations. For these cases, the program will be unable to distinguish between an amino acid or nucloetide sequence--this however can be corrected by putting a "*" at the end of an amino acid sequence.
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| − | <div class="row">
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| − | <div class="col-sm-12 pagetext">
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| − | <ol>
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| − | <li>First sequence contains at least 50nt of the 3' end of the backbone where the 5' end of the first fragment will join to</li>
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| − | <li>N number of sequences to be assembled ...</li>
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| − | <li>Last sequence contains the 5' end of the backbone where the the 3' end of the last fragment will join to This is illustrated in the following figure, where the first sequence in the file is "Backbone front", last sequence is "Backbone rear", and the middle sequences in the file would be the N fragments listed in order of assembly.</li>
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| − | </ol>
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| | <div class="col-sm-12 imgcol-L"> | | <div class="col-sm-12 imgcol-L"> |
| | <img src="https://static.igem.org/mediawiki/2016/2/20/T--Stanford-Brown--ProtOpt_Frags.png" class="img-L"/> | | <img src="https://static.igem.org/mediawiki/2016/2/20/T--Stanford-Brown--ProtOpt_Frags.png" class="img-L"/> |
