Team:UCLA/Notebook/Protocols

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    <div class= "content_subsection" id="protocols">
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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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<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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<a href="https://drive.google.com/file/d/0B8rwOFG2xrHdX2xadGdMdzgxMjA/view?usp=sharing">Customizing & Functionalizing Silk Project Notebook</a>
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          <h2>Processing Silk</h2>
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          <p>This team's focus was to hammer out procedures for producing and processing various silk-based materials. This includes working with <i>Bombyx mori</i> silk to produce films, and gels, as well as designing the Extrusion Column and Rotary Jet Spinner.</p>
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<a href="https://drive.google.com/file/d/0B8rwOFG2xrHdSHk0VnFNY1lnbDQ/view?usp=sharing">Processing Silk Project Notebook</a>
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Revision as of 23:34, 17 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
Customizing & Functionalizing Silk Project Notebook

Processing Silk

This team's focus was to hammer out procedures for producing and processing various silk-based materials. This includes working with Bombyx mori silk to produce films, and gels, as well as designing the Extrusion Column and Rotary Jet Spinner.

Processing Silk Project Notebook

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 silk
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.