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Team:UCLA/Notebook/Protocols
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| - | + | <h1>Protocols</h1> | |
| - | + | <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/> | <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/> | ||
<a href="https://static.igem.org/mediawiki/2014/a/ab/Equipment_Protocol.pdf">Equipment for Recombinant Silk Production</a><br/> | <a href="https://static.igem.org/mediawiki/2014/a/ab/Equipment_Protocol.pdf">Equipment for Recombinant Silk Production</a><br/> | ||
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<li>Amino Acid Analysis</li> | <li>Amino Acid Analysis</li> | ||
<li>Overall: Timing of Procedure</li> | <li>Overall: Timing of Procedure</li> | ||
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| - | < | + | <h2>Iterative Capped Assembly</h2> |
<a href="https://static.igem.org/mediawiki/2014/b/b6/ICA_Protocol_Paper.pdf">Protocol Paper for Iterative Capped Assembly</a><br/> | <a href="https://static.igem.org/mediawiki/2014/b/b6/ICA_Protocol_Paper.pdf">Protocol Paper for Iterative Capped Assembly</a><br/> | ||
<br>Outline</br> | <br>Outline</br> | ||
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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> | <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> | ||
<li>Ligate purified product into backbone using restriction sites on the prefix and suffix</li> | <li>Ligate purified product into backbone using restriction sites on the prefix and suffix</li> | ||
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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/> | <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/> | ||
<br>Outline</br> | <br>Outline</br> | ||
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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> | |
<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> | <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> | ||
<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." </span> <em>Nucleic acids research</em><span> (2012): gks624.</br> | <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." </span> <em>Nucleic acids research</em><span> (2012): gks624.</br> | ||
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Latest revision as of 01:01, 18 October 2014
Protocols
Recombinant Silk Proteins using Head to Tail Method
Protocol Paper for Producing Recombinant Silk with Head to Tail MethodEquipment for Recombinant Silk Production
Reagent List for Recombinant Silk Production
Outline
- Construction of the Synthetic Spider Silk Multimeric Gene
- Cloning of the spider silk multimer into the expression vector
- Gene Expression of pETsilk
- E.coli Cell Lysis
- Silk Protein Purification Using IMAC
- Dialysis
- Protein Analysis
- Amino Acid Analysis
- Overall: Timing of Procedure
Iterative Capped Assembly
Protocol Paper for Iterative Capped AssemblyOutline
- Assemble monomers via PCR and prepare all other oligos, i.e. the initiator, terminator, and capping oligos
- Add biotinylated initiator oligo to streptavidin coated beads
- 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
- After spider silk sequence has the desired number of repeats, add terminator oligo and elute DNA from beads by heating the reaction
- 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
- 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 MaterialsOutline
- Dissolve silk in LiBr Solution
- Dialyze silk solution against ultrapure water to remove salts
- Dialyzed product will be light yellow and slightly viscous- can be stored in 4° C fridge for about a month
- For silk film: pour thin layer of dialyzed silk into plate and let dry overnight
- For silk pH gels: make a 1 HCl:10 dialyzed silk solution using 1M HCl, and let solidify overnight
- For silk fibers:
- Option 1: Lyophilize dialyzed silk for 2-3 days. Resuspend lyophilized silk powder in HFIP, and extrude through syringe
- Option 2: Redialyze silk solution once more against Polyethylene Glycol (PEG) to concentrate it. Extrude concentrated silk through syringe
Reference
Materials FabricationRockwood, 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 SilkTeulé, 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 AssemblyBriggs, Adrian W., et al. "Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers."  Nucleic acids research (2012): gks624.
