Team:UTP-Software/SoftwareTool

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In some cases, it’s impossible to fix some sequences, because based on the protocol, the output primer may not comply with the steps that are listed in the “Protocol Followed to design the primers” section. Meaning that another standard should be selected.  </ul></div>
In some cases, it’s impossible to fix some sequences, because based on the protocol, the output primer may not comply with the steps that are listed in the “Protocol Followed to design the primers” section. Meaning that another standard should be selected.  </ul></div>
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==== Protocol Followed to design the primers:<sup>[1]</sup> ====
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==== Flow diagram for the S<sup>2</sup>MT - Primer Design Section ====
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[[File:Flow diagram.png|center]]
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==== Protocol Followed to design the primers:<sup>[http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf]</sup> ====
<div align="justify"><ul>
<div align="justify"><ul>
<li> Both the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.</li>
<li> Both the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.</li>
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<li> Primers need not be 5´ phosphorylated but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency.</li>
<li> Primers need not be 5´ phosphorylated but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency.</li>
<li> It is important to keep primer concentration in excess. Stratagene suggests varying the amount of template while keeping the concentration of the primer constantly in excess.</li></ul></div>
<li> It is important to keep primer concentration in excess. Stratagene suggests varying the amount of template while keeping the concentration of the primer constantly in excess.</li></ul></div>
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<div align="center"><ul>
 
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[[File:Flow diagram.png]]
 
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Flow diagram for the S<sup>2</sup>MT
 
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</ul></div>
 
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== '''S<sup>2</sup>MT Tutorial''' ==
 
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<div align="justify"><ul>
 
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[[File:Img1.png]]
 
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(S<sup>2</sup>MT) GUI parts:
 
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<li>1. URL/DNA Sequence textbox:  This is the main input.</li>
 
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<li>2. Standard selection: The assembly reference we want the script take as the desired assembly standard and check the sequence compatibility with, fixing it if needed.</li>
 
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<li>3. Process button: After giving the inputs, analyze the sequence.</li>
 
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<li>4. Primer textbox: Here we get the output.</li>
 
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<li>5. Notification area: The script's feedback.
 
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</li></ul></div>
 
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<div align="justify"><ul>
 
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Some points to take in consideration:
 
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<li>The primer's starting positions will appear in the MATLAB's console.
 
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<li>The sequence box can either get an URL from the Parts Registry pointing to a specific Part or a DNA sequence (the textbox is case insensitive).</li>
 
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<li>Some PartsRegistry Devices does not have it corresponding DNA Sequence on it.</li>
 
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<li>Be sure that the DNA Sequence you put in the box does not contain any other letter than 'a', 't', 'g' and 'c'.</li>
 
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<li>Some sequence's incompatibility can't be fixed.
 
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</li></ul></div>
 
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<div align="justify"><ul>
 
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[[File:Fix.png]]
 
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(S<sup>2</sup>MT) succesfully fixing the incompatibility of the given DNA sequence with the standard 21.
 
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To download the script go to: https://github.com/igemsoftware/UTP_Software_2012 and get all three files.
 
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Files descriptions:
 
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<li>S2MT.m: Does all the sequence analysis process.</li>
 
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<li>S2MT_G.m: Code for the grafical interface. (Run this)</li>
 
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<li>S2MT_G.fig: Binary file for the grafical interface.</li>
 
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Note that (S<sup>2</sup>MT) relies on the Bioinformatics Toolbox of MATLAB (both supplied by iGEM) to make its computations, so you need the toolbox to be able to run the script.
 
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</ul></div>
 
== Next S<sup>2</sup>MT Version ==
== Next S<sup>2</sup>MT Version ==
<div align="justify">After finishing the first version, we had in mind several things to implement to the next:  
<div align="justify">After finishing the first version, we had in mind several things to implement to the next:  
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<ul><li>Regarding assembly standards which was our first main idea, we think that the actual 5 available standards accepted by the part registry may not always be enough for the teams to manifest their ideas, because in some cases even with our Site directed mutagenesis method, fixing the sequence may fail or be impossible to do. Then keeping this in mind, we would like to add more assembly standards to the competition, and add those new to our system.</li>
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<ul><li> Our next version will for sure incorporate a "ab1" chromatogram file reader, because right now we are relying on phred files, which eventhoug phred scores are pretty accurate, not every sequencing company provide those.
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One of them may be the Gibson Assembly standard, which still requires compatibility with the actual methods, but include several advantages as:
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Some bugs are still to be fixed, and some performance improvement can also be a part of the next version. 
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#No restriction digest of the DNA fragments after PCR is necessary. The backbone vector can be digested, or synthesized by PCR.
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#It is far simpler than conventional cloning schemes, as it requires fewer steps and fewer reagents. The process also takes less time.
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<li>Our Second idea (still under development) is about adding a whole new tool to our software, and thats what we call BioSinergia (spanish for "BioSinergy"), which will be like a smart database of metabolic rutes that is going to help teams and researchers to work and study the production of bioenergy through synthetic biology. This project will make use of our S<sup>2</sup>MT tool to make it easy for new iGEM teams to work with biofuels in their projects.
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#No restriction site scar remains between two DNA fragments.
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#Multiple DNA fragments can be combined simultaneously in a single reaction.
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</ul></div>
</ul></div>
== References ==
== References ==
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[1]. <b>Stratagene</b>. QuikChange™ Site-Directed Mutagenesis Kit. Catalog #200518. Revision #108005h. Available in:
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[1]. <b>Stratagene</b>. QuikChange™ Site-Directed Mutagenesis Kit. Catalog #200518. Revision #108005h. Available in: http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf.
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''[http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf]''.
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[2]. Prather:Gibson CBA. Available in: http://openwetware.org/wiki/Prather:Gibson_CBA

Latest revision as of 03:47, 27 October 2012

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Project Details

What is the S2MT tool?

    The SynBio Sequence Mutation Tool (S2MT) it's a MATLAB implementation software that helps teams to start their projects once they have selected some possible BioBricks or DNA sequences to work with.

    This tool was developed in MATLAB basically for the reason that, what new iGEM teams require is a flexible environment capable of not only running the algorithm that we develop, but also a place where they can continue working their ideas, examine more in detail their sequences and even continue the development of our tool. So we thing that our system will also knock down the barriers that exist between computation and biology and give students the tools required to do better simulations, and save a lot of valuable time!

    Behind the code there are three simple steps in which the program consists:

  • First, the user introduces the DNA sequence or link to the BioBrick in the part registry. And the program scans the desired sequence to look for the restriction sites within the assembly standards that are used in the iGEM competition.
  • Then the user is prompt to specify in case of incompatibility with the standards, which RFC he would like to fix. Once selected, the program designs the primers required for the Site directed mutagenesis procedure that will allow the sequence to be compatible with the standard.
  • And last, if the program successfully created the required primer, it will print the sequence, giving the user the start position where that primer should be applied.
    • In some cases, it’s impossible to fix some sequences, because based on the protocol, the output primer may not comply with the steps that are listed in the “Protocol Followed to design the primers” section. Meaning that another standard should be selected.

Flow diagram for the S2MT - Primer Design Section

Flow diagram.png

Protocol Followed to design the primers:[http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf]

  • Both the mutagenic primers must contain the desired mutation and anneal to the same sequence on opposite strands of the plasmid.
  • Primers should be between 25 and 45 bases in length, and the melting temperature (Tm) of the primers should be greater than or equal to 78°C. The following formula is commonly used for estimating the Tm of primers:

    • Tm = 81.5 + 0.41(%GC) − 675 / N - where N is the primer length in base pairs.

  • The desired mutation (deletion or insertion) should be in the middle of the primer with ~10–15 bases of correct sequence on both sides.
  • The primers optimally should have a minimum GC content of 40% and should terminate in one or more C or G bases.
  • Primers need not be 5´ phosphorylated but must be purified either by fast polynucleotide liquid chromatography (FPLC) or by polyacrylamide gel electrophoresis (PAGE). Failure to purify the primers results in a significant decrease in mutation efficiency.
  • It is important to keep primer concentration in excess. Stratagene suggests varying the amount of template while keeping the concentration of the primer constantly in excess.

Next S2MT Version

After finishing the first version, we had in mind several things to implement to the next:
  • Our next version will for sure incorporate a "ab1" chromatogram file reader, because right now we are relying on phred files, which eventhoug phred scores are pretty accurate, not every sequencing company provide those. Some bugs are still to be fixed, and some performance improvement can also be a part of the next version.
  • Our Second idea (still under development) is about adding a whole new tool to our software, and thats what we call BioSinergia (spanish for "BioSinergy"), which will be like a smart database of metabolic rutes that is going to help teams and researchers to work and study the production of bioenergy through synthetic biology. This project will make use of our S2MT tool to make it easy for new iGEM teams to work with biofuels in their projects.

References

[1]. Stratagene. QuikChange™ Site-Directed Mutagenesis Kit. Catalog #200518. Revision #108005h. Available in: http://web.physics.ucsb.edu/~deborah/pro/pro_pdf/Stratagene%20QuikChange.pdf.

[2]. Prather:Gibson CBA. Available in: http://openwetware.org/wiki/Prather:Gibson_CBA

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