Team:UCLA/Notebook/Spider Silk Genetics/Protocols/Iterative Capped Assembly

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This document details steps to perform ICA for Spider Silk Gene Assembly.

ICA Preparation

• Required Solutions:
  • 2x BW Buffer
  • 0.5x BW Buffer
  • Elution Buffer
• Prepared, working solutions of the Initator, Terminator and Capping Oligos
• Purified, BsaI Digested MaSp Monomers
  • Sufficient amount to complete ICA for the desired chain length.
  • 50 ng monomer is used in each extension reaction.

Iterative Capped Assembly

1. Resuspend the M-270 Streptavidin beads by gently shaking the bottle.
2. Aliquot 5 uL of beads into tube. Apply the magnetic strip to isolate the beads.
   • Wash twice with 100 uL of 2x BW Buffer
3. Resuspend the beads in 5 uL 2x BW Buffer, 4 uL ddH2O, and 1 uL 50 nM Initiator.
   • Incubate on a rotator at RT for 45 minutes.
   • This incubation period can be used to prepare the subsequent ligation reactions ahead of time. Mix all the relevant reagents together EXCEPT the T7 Ligase in separate tubes, one tube per reaction.
4. Apply the magnetic strip to isolate the beads and use a pipette to remove residual solution. (All future wash steps require the magnetic strip to isolate the beads)
   • Wash twice with 100 uL of 0.5x BW Buffer.
5. Add in the components of the first ligation:
   • 5 uL 2x T7 Ligase Buffer
   • 50 ng of piece AB
   • ddH2O to 9.5 uL
6. Add 0.5 uL of T7 Ligase (temperature sensitive, put back in freezer between use)
7. Completely resuspend beads using pipet or by gently flicking.
8. Incubate on rotator at RT for 3 min.
9. Place on magnetic rack and remove residual solution.
10. Wash 2x using 100 uL 0.5x BW Buffer
11. Mix in components of second ligation:
    • 5 uL 2x T7 Ligase Buffer
    • 50 ng of piece BC
    • 1 uL of 5 uM A-cap
    • ddH2O to 9.5 uL
12. Add 0.5 uL T7 Ligase and resuspend completely.
13. Incubate on rotator at RT for 3 min.
14. Repeat wash and ligation steps as many times as needed to assemble the construct.
15. For the final ligation mix the following:
    • 5 uL 2x T7 Ligase Buffer
    • 1 uL of 5 uM working Terminator oligo
    • 1 uL of C-cap
    • 2.5 uL ddH2O.
16. Resuspend the beads and add 0.5 uL T7 Ligase Buffer.
17. Incubate on rotator for 3 min.
18. Remove residual solution.
19. Wash once using 100 uL 0.5x BW Buffer.
20. Wash once using 100 uL ddH2O.
21. Resuspend beads in 15 uL of ICA Elution Buffer.
22. Incubate at 95 C for 5 min.
23. Apply magnetic strip and save the solution in an appropriately labeled tube.

ICA Considerations

• The part collection we designed for ICA with spider silk genes contains two parts indicated as MaSp1 SeqAB and MaSp2 SeqAB. These parts have a different DNA sequence than the other MaSp1 and 2 pieces respectively, but code for the same protein. These parts assist in sequencing long ICA constructs.

ICA Product Amplification and cloning

• Use one microliter of the ICA eluate as template for PCR amplification using post-elution primers.
• The amplified product can be purified and cloned into the vector of choice.