IGEM:Harvard/2006/DNA nanostructures/Notebook/2006-7-18

Questions

Design 2

• How big does a 2-ply lid need to be to span a 30-helix barrel?
  • There are two estimated diameters for double-ply helices, 1.82nm and 2nm. These give, respectively, lengths of 28.6nm or 31.4nm for each 18-helix-long double-ply lid (36 helices in total). This model uses 3456 bp each, 6912 bp together if both lids are coded on one scaffold, and with the necessary spacer between lids to be as long as the barrel (ie. +84bp), 7080 bp, and if the 200bp spacer between lid and barrels are designed in, 7480bp total.
  • Since the 30-helix single-ply barrel is 28.9nm at its widest point, a longer double-ply lid may be needed.
  • Another possibility is making each lid out of its own scaffold, which would allow each to be coded with different sections of the p7308 M13 phage genome. The 30-helix single-ply barrel only require 2880 bp, which would allow it to be coded on the phiX174 genome (5386bp long).

• Can we use two scaffolds in the design?
  • 2x42bp repeats in barrel
  • 30-helix bundle with 30*(2*42+12) repeats = 2880 bases
  • two-ply-lid: 36 * (2*42+12) = 3456 * 2 = 6912
  • using single scaffold for 2880-base 30hb: 9792 bases total = $783.36
  • 3x42bp repeats in barrel
  • 30-helix bundle with 30*(3*42+12) repeats = 4140 bases
  • two-ply-lid: 36 * (2*42+12) = 3456 * 2 = 6912
  • using single scaffold for 4140-base 30hb: 11052 bases total = $884.16

• Will unused scaffold interfere with proper folding of lid & barrels?
• Should we use two M13-based scaffolds, or try to make ΦX174 to have less slack?
• Can we easily get ΦX174? fermentas

Protection assay

Questions

• Can we digest streptavidin with Qiagen proteinase K and/or Qiagen protease?
• What alternative proteases can we use?
  • We're going to try trypsin
• What is our initial protocol?

Proteinase K protocol

• Wu's digestion protocol [1]: Proteinase K Digestion of Streptavidin and Its Muteins—Purified streptavidin and its muteins (30 μM monomer) were treated with proteinase K (Invitrogen, 5 μM) for 15 min at 30[[:Category:{{{1}}}|{{{1}}}]] in 50 mM Tris-HCl containing 5 mM CaCl2, pH 8.0. The reaction was stopped by precipitation with trichloroacetic acid (18). Boiled samples of precipitated proteins were resolved by reducing SDS-PAGE. The same analysis was performed with streptavidin samples treated with biotin (1 mM final concentration) prior to proteinase K digestion.
• A possible protocol:
  • Streptavidin tetramer (20 μM) is incubated with 3'-biotinylated oligo (10 μM) for 5 min. at room temperature (28[[:Category:{{{1}}}|{{{1}}}]] today, whew)
  • Streptavidin tetramer (one expt. biotin-treated, one expt. biotin-untreated), is treated with 5 μM proteinase K for 15 min at 30[[:Category:{{{1}}}|{{{1}}}]] in 50 mM Tris-HCl containing 5 mM CaCl2, pH 8.0
  • Reaction is stopped by EtOH precipitation of DNA, or by filtration through a Qiagen column