Team:UCLA/Notebook/Protocols

Protocols

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

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

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