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 make a double-ply barrel (42-helix=outer barrel, 30-helix=inner barrel)?
  • A double-ply helix can be made with (42+30)-1 helices, taking 6816 bp, requiring the use of the M13 phage genome.
  • If the diameter of the helices are assumed to be the two estimated diameters found with the double-ply pleated sheets (ie. 1.82nm or 2nm), the barrel is 23.9nm or 26.2nm wide at its widest point. This allows the use of 18-helix-long double-ply lids (36 helices in total), which are estimated 28.6 or 31.4nm long.
  • If the normal 3nm diameter is assumed, the double-ply barrel will be 39.4nm at its widest point, necessitating a 24-helix-long lid (48 helices in total), or 4608bp per lid. This would require each lid be coded in its own scaffold, and would also require using overlapping parts of that scaffold sequence to code the lids.
  • However, its most likely that the barrel is between 31.4nm and 39.4nm at its widest point. A 20-helix-long lid (40 helices in total), or 3840bp per lid, could likely be used. This would require overlapping of the parts of the scaffold sequence to be used, since phiX174 genome is necessary, as M13 will be used for the double-ply barrel.

• 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

Container 3.2

Pre-working stocks

• Mix 2.5 μL of each 200 μM stock oligo (add 7.5 μL H₂O per oligo to dilute to 50 μM)

pre-working stock	desc	oligos plate locations	total
c3.2.6b	barrel at inside aptamer locations +3'biotinaptamers	3.2.6.1b, .2b, .3b	3
c3.2.7b	barrel at inside aptamer locations +3'biotinaptamers	3.2.7.1b, .2b, .3b	3

Working stocks

Final concentration of each oligo in working stocks is 250 nM.

working stock	description	pre-working stocks (according to chart)	water	total
c3.2.Ib.core	+latch2 +biotinaptamers out	1 (94 μL), 2 (28 μL), 3 (30 μL), 4 (3 μL), 7b (3 μL), 10 (2 μL), 12 (2 μL), 13 (0 μL), 15 (2 μL)	36 μL	200 μL
c3.2.Ib.latches	+latch2 +biotinaptamers out	16 (4 μL)	-	-