Team:UCLA/Notebook/Protocols

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<p>You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well. </p>
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          <h1>Protocols</h1>
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          <h2>Recombinant Silk Proteins using Head to Tail Method</h2>
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<a href="https://static.igem.org/mediawiki/2014/6/66/Recombinant_Silk_Protocol_Paper.pdf">Protocol Paper for Producing Recombinant Silk with Head to Tail Method</a><br/>
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<a href="https://static.igem.org/mediawiki/2014/a/ab/Equipment_Protocol.pdf">Equipment for Recombinant Silk Production</a><br/>
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<a href="https://static.igem.org/mediawiki/2014/9/92/Reagent_Protocol.pdf">Reagent List for Recombinant Silk Production</a><br/>
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<br>Outline</br>
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<ol>
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<li>Construction of the Synthetic Spider Silk Multimeric Gene</li>
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<li>Cloning of the spider silk multimer into the expression vector</li>
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<li>Gene Expression of pETsilk</li>
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<li>E.coli Cell Lysis</li>
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<li>Silk Protein Purification Using IMAC</li>
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<li>Dialysis</li>
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<li>Protein Analysis</li>
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<li>Amino Acid Analysis</li>
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<li>Overall: Timing of Procedure</li>
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</ol>
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          <h2>Iterative Capped Assembly</h2>
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<a href="https://static.igem.org/mediawiki/2014/b/b6/ICA_Protocol_Paper.pdf">Protocol Paper for Iterative Capped Assembly</a><br/>
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<br>Outline</br>
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<ol>
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<li>Assemble monomers via PCR and prepare all other oligos, i.e. the initiator, terminator, and capping oligos</li>
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<li>Add biotinylated initiator oligo to streptavidin coated beads</li>
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<li>Add monomers in desired sequence with T7 DNA ligase, making sure to add blocks with AB, BC, and CA overhangs sequentially. Incorporate a wash step and capping step after adding each monomer</li>
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<li>After spider silk sequence has the desired number of repeats, add terminator oligo and elute DNA from beads by heating the reaction</li>
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<li>Amplify eluted DNA by PCR with primers that are complementary to the biobrick prefix on the ICA initiator, and the biobrick suffix on the ICA terminator</li>
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<li>Ligate purified product into backbone using restriction sites on the prefix and suffix</li>
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</ol>
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          <h2>Materials Fabrication from <i>Bombyx mori</i></h2>
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<a href="https://static.igem.org/mediawiki/2014/4/4e/Silk_Materials_Protocol_Paper.pdf">Protocol Paper for Producing Various Silk-based Materials</a><br/>
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<br>Outline</br>
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<ol>
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<li>Dissolve silk in LiBr Solution</li>
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<li>Dialyze silk solution against ultrapure water to remove salts</li>
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<li>Dialyzed product will be light yellow and slightly viscous- can be stored in 4&deg C fridge for about a month</li>
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<li>For silk film: pour thin layer of dialyzed silk into plate and let dry overnight</li>
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<li>For silk pH gels: make a 1 HCl:10 dialyzed silk solution using 1M HCl, and let solidify overnight</li>
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<li>For silk fibers:
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  <ol>
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      <li>Option 1: Lyophilize dialyzed silk for 2-3 days. Resuspend lyophilized silk powder in HFIP, and extrude through syringe</li>
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      <li>Option 2: Redialyze silk solution once more against Polyethylene Glycol (PEG) to concentrate it. Extrude concentrated silk through syringe</li>
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  </ol>
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</li>
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</ol>
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<h3> Reference </h3><b>Materials Fabrication</b></br>Rockwood, Danielle N., Rucsanda C. Preda, Tuna Yücel, Xiaoqin Wang, Michael L. Lovett, and David L. Kaplan. "Materials Fabrication from Bombyx Mori Silk Fibroin." Nature Protocols 6.10 (2011): 1612-631. Web. <br>
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<br><b>Production of Recombinant Silk</b></br>Teulé, Florence, Alyssa R. Cooper, William A. Furin, Daniela Bittencourt, Elibio L. Rech, Amanda Brooks, and Randolph V. Lewis. "A Protocol for the Production of Recombinant Spider Silk-like Proteins for Artificial Fiber Spinning." Nature Protocols 4.3 (2009): 341-55. Web.</br>
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<br><b>Iterative Capped Assembly</b></br>Briggs, Adrian W., et al. "Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers."&nbsp</span> <em>Nucleic acids research</em><span>&nbsp(2012): gks624.</br>
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Latest revision as of 01:01, 18 October 2014

iGEM UCLA





Protocols

Recombinant Silk Proteins using Head to Tail Method

Protocol Paper for Producing Recombinant Silk with Head to Tail Method
Equipment for Recombinant Silk Production
Reagent List for Recombinant Silk Production

Outline
  1. Construction of the Synthetic Spider Silk Multimeric Gene
  2. Cloning of the spider silk multimer into the expression vector
  3. Gene Expression of pETsilk
  4. E.coli Cell Lysis
  5. Silk Protein Purification Using IMAC
  6. Dialysis
  7. Protein Analysis
  8. Amino Acid Analysis
  9. Overall: Timing of Procedure



Iterative Capped Assembly

Protocol Paper for Iterative Capped Assembly

Outline
  1. Assemble monomers via PCR and prepare all other oligos, i.e. the initiator, terminator, and capping oligos
  2. Add biotinylated initiator oligo to streptavidin coated beads
  3. Add monomers in desired sequence with T7 DNA ligase, making sure to add blocks with AB, BC, and CA overhangs sequentially. Incorporate a wash step and capping step after adding each monomer
  4. After spider silk sequence has the desired number of repeats, add terminator oligo and elute DNA from beads by heating the reaction
  5. Amplify eluted DNA by PCR with primers that are complementary to the biobrick prefix on the ICA initiator, and the biobrick suffix on the ICA terminator
  6. Ligate purified product into backbone using restriction sites on the prefix and suffix



Materials Fabrication from Bombyx mori

Protocol Paper for Producing Various Silk-based Materials

Outline
  1. Dissolve silk in LiBr Solution
  2. Dialyze silk solution against ultrapure water to remove salts
  3. Dialyzed product will be light yellow and slightly viscous- can be stored in 4&deg C fridge for about a month
  4. For silk film: pour thin layer of dialyzed silk into plate and let dry overnight
  5. For silk pH gels: make a 1 HCl:10 dialyzed silk solution using 1M HCl, and let solidify overnight
  6. For silk fibers:
    1. Option 1: Lyophilize dialyzed silk for 2-3 days. Resuspend lyophilized silk powder in HFIP, and extrude through syringe
    2. Option 2: Redialyze silk solution once more against Polyethylene Glycol (PEG) to concentrate it. Extrude concentrated silk through syringe



Reference

Materials Fabrication
Rockwood, Danielle N., Rucsanda C. Preda, Tuna Yücel, Xiaoqin Wang, Michael L. Lovett, and David L. Kaplan. "Materials Fabrication from Bombyx Mori Silk Fibroin." Nature Protocols 6.10 (2011): 1612-631. Web.

Production of Recombinant Silk
Teulé, Florence, Alyssa R. Cooper, William A. Furin, Daniela Bittencourt, Elibio L. Rech, Amanda Brooks, and Randolph V. Lewis. "A Protocol for the Production of Recombinant Spider Silk-like Proteins for Artificial Fiber Spinning." Nature Protocols 4.3 (2009): 341-55. Web.

Iterative Capped Assembly
Briggs, Adrian W., et al. "Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers."&nbsp Nucleic acids research&nbsp(2012): gks624.

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