User:Matthewmeisel

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Notebook

Week 2

Tue Jun 20

Transformation of standard components

• cultured bacteria did grow overnight
• 1 mL of reserved to make glycerol stock:

1. 666 μL of cells and medium and 666 μL of 50% glycerol solution in water
2. stored at 80[[:Category:{{{1}}}|{{{1}}}]]

• extracted DNA from remaining 4 mL using the standard protocol:

1. Cells and medium poured into several 1.5 mL microcentrifuge tubes, centrifuged, and supernatant discarded.
2. Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 °C).
3. Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.
4. Added 350 μl Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.
5. Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.
6. Combined the supernatants from step 4 in a single QIAprep spin column by decanting.
7. Centrifuged for 60 s. Discarded the flow-through.
8. Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.
9. Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.
10. Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.

Mon Jun 19

Transformation of standard components

• Viewed plate from Thu, about a dozen colonies were flourescing brightly
• Selected one colony for amplification:

1. 5 mL LB medium, 50 μL of 5 mg/mL ampicillin, and one colony into a culture tube
2. Shaken at 18 h at 37[[:Category:{{{1}}}|{{{1}}}]]

Week 1

Thu Jun 15

Transformation of standard components

• ligation and transformation of promoter and GFP
• ligation protocol with the Roche Rapid DNA ligation kit:

1. 6 μL of insert (E0241), 1 μL of plasmid (R0010 and backbone), and 3 μL of 1x DNA dilution buffer (vial 2) (enough buffer for total volume of 10 μL) into a microcentrifuge tube. (Would ordinarily use 2 μL of plasmid, but the plasmid concentration is about doubled because it combined bands from two gel lanes.)
2. Mixed ligation buffer (vial 1), and added 10 μL to tube.
3. Added 1 μL T4 DNA ligase (vial 3).
4. Incubated on ice for 5 min.

• transformation:

1. 30 μL competent cells each into three microcentrifuge tubes.
2. Ligation mixture, 1 μL water (negative control), and 1 μL pUC 19 (positive control) added to respective tubes.
3. Incubated the tubes on ice for 20 min.
4. Heat shocked the tubes at 42°C for 30 s.
5. Added 200 μL SOC to each tube.
6. Incubated, with shaking, at 37°C for 1 h.
7. Plated the mixture on ampicillin plates and incubated at 37°C for 24 h, then stored at 4°C.

Wed Jun 14

Transformation of standard components

• cultured bacteria did grow overnight
• 1 mL of each tube reserved to make glycerol stock:

1. 666 μL of cells and medium and 666 μL of 50% glycerol solution in water
2. stored at 80[[:Category:{{{1}}}|{{{1}}}]]

• extracted DNA from remaining 4 mL using the standard protocol:

1. Cells and medium poured into 1.5 mL microcentrifuge tubes, centrifuged, and supernatant discarded.
2. Resuspended pelleted bacterial cells in 250 µl Buffer P1 (w/ RNAse) (kept at 4 °C) and transfered to a microcentrifuge tube.
3. Added 250 μl Buffer P2 and gently inverted the tube 4–6 times to mix. Waited 2 min.
4. Added 350 μl Buffer N3 and inverted the tube immediately but gently 4–6 times. Solution became cloudy.
5. Centrifuged for 10 min at 14,000 rpm. A compact white pellet formed.
6. Applied the supernatants from step 4 to the QIAprep spin column by decanting.
7. Centrifuged for 60 s. Discarded the flow-through.
8. Washed the QIAprep spin column with 0.5 ml Buffer PB and centrifuged for 60 s. Discarded the flow-through.
9. Washed QIAprep spin column by with 0.75 ml Buffer PE and centrifuged for 60 s.
10. Placed the QIAprep column in a clean 1.5 ml microcentrifuge tube. Eluted with 30 μl water to the center of each QIAprep spin column, let stand for 2 min, and centrifuged for 60 s.

• digested promoter (R0010) and GFP (E0241) plasmids
  • R0010 digested with SpeI and PstI in order to leave it attached at upstream end to the plasmid backbone (otherwise, the fragment would only be ~200 bp long, which is a little too short for electrophoresis with much longer fragments
  • E0241 digested with XbaI and PstI in order to cleave it as a fragment

1. The following ingredients were each added to four 1.5 ml centrifuge tubes:
   • 10 μl water
   • 8 μl DNA from the previous step (R0010-1, R0010-2, E0241-1, E0241-2 in respective tubes)
   • 2.5 μl 10x NEB buffer (#2 for R0010-01 and R0010-2, #3 for E0241-1 and E0241-2)
   • 2.5 μl 10x BSE
   • 1.0 μl 1:1 diluted enzyme A (SpeI for R0010-1 and R0010-2, XbaI for E0241-1 and E0241-2)
   • 1.0 μl 1:1 diluted enzyme B (PstI for all).
2. Tubes were incubated at 37°C for 90 min.
3. Tubes were incubated at 80°C for 15 min in order to inactivate the enzymes.
4. All four samples were run on a 1% agarose gel:

Lane	Contents	Loading Buffer
0	1kb DNA ladder (10 μL)	10x loading dye (1.1 μL)
1	E0241-1 (15 μL)	10x loading dye (2.5 μL)
2	E0241-2 (15 μL)	10x loading dye (2.5 μL)
3	R0010-1 (15 μL)	10x loading dye (2.5 μL)
4	R0010-2 (15 μL)	10x loading dye (2.5 μL)

Tue Jun 13

DNA nanostructure reaction PCR

• order of lanes: 1) 1kb ladder, 2) full rxn (oligos + scaffold), 3) just scaffold, 4) just oligos
• run on 2% agarose gel
• appears that reaction was successful: gel image (leftmost four lanes) shows expected results
  • assembled nanostructure runs slightly faster than scaffold, oligos appear as a smear of short DNAs

Transformation of standard components

• results of plated bacteria: all three plates showed colony growth, negative control did not
• colony selection and amplification

1. Seven culture tubes were filled with 5 mL LB medium and 50 μl of 5 mg/mL ampicillin
2. Colonies were selected from the three plates (three from R0010, two from E7104, two from E0241) and were transferred into respective culture tubes with a sterilized pick
3. Culture tubes were incubated, with shaking, at 37°C for 18 h.
4. Plates were stored at 4°C.

Mon Jun 12

DNA nanostructure reaction PCR

• standard protocol with materials from Shawn

Transformation of standard components

• goal: insert three BioBrick plasmids (already containing BioBricks) into E. coli in order to amplify them
  • a positive control (E7104) with the T7 promoter upstream of GFP
  • the lac operon (R0010)
  • GFP (E0241)
• transformation protocol:

1. 1 μL each of E7104, R0010, E0241, and water into separate microcentrifuge tubes that each contain 30 μL of competent cells
2. Incubated the tubes on ice for 20 min.
3. Heat shocked the tubes at 42°C for 30 s.
4. Added 200 μL SOC to each tube.
5. Incubated, with shaking, at 37°C for 1 h.
6. Plated the mixture on ampicillin plates and incubated at 37°C for 24 h.

Literature summaries and brainstorming

Shih's 2004 Nature paper (biblio below)

Shih describes the (rather complex) construction of the DNA octahedron, which consists of two motifs: double-crossover edges ("struts") and paranemic crossover struts. Each edge of the octahedron is combrised of two ds DNAs which are interwoven with these motifs. Edges are connected at vertices by two unpaired thymine residues for flexibility. The structure was formed from a long "heavy chain" (scaffold) and five 40-nt "light chains" (oligos) in two steps: first, by cooling the structure from a denaturation temperatue to form a branched complex, and then further cooling to allow corresponding paranemic crossover areas to associate.

The final formation step is proven through gel electrophoresis. Mg²⁺ is required for the final folding step, and the product runs faster after Mg²⁺ has been added, indicating further folding. Other confirmatory steps were complicated: the structures were visualized with cryo-electron microscopy, and a 3d model was formed using microscopy images from 961 particles.

Shih points out that no covalent bonds are created or broken in the formation of the structure, which greatly simplifies the assembly process. He also suggests one possible application/implication of his work, which is that one-dimensional information (the primary DNA sequence of the heavy chain) is effectively encoding 3d positional information (i.e., the final 3d position of the nucleotide).

Yurke's 2000 Nature paper (biblio below)

Yurke's diagram of his DNA tweezers

Yurke describes the DNA "tweezers" he engineered, which consist of the following parts:

• a short (~30 nt) ss DNA backbone (A)
• two oligos (B and C) which are each complimentary to half of the backbone and bound to it, and each of which have a short (~20 nt) ss overhang

In this state, the DNA tweezers are in an "open" conformation. A ~50-nt "fuel strand" of ss DNA (F) is added to the tweezers to close them, and this occurs because the fuel strand is complimentary to each of the oligo overhangs, which closes the tweezers. The fuel strand also includes a 8-nt ss overhang, and so when a strand complimentary to the fuel strand ("displacement" strand") (F-bar) is added, the fuel strand is stripped, opening the tweezers.

Yurke reports that the rate-limiting step of this "strand displacement" is the initial binding between the fuel strand and the displacement strand, and the time required for DNA branch migration (on this scale) is negligble. The tweezers open and shut on a time scale of a few minutes after a fuel (or displacement) strand is added.

Link to Animation

Thoughts on Shih and Yurke

A convenient way to open a DNA nanobox would be to construct a box held shut by a latch with a DNA tweezer motif in its closed conformation, such that a displacement strand would open the box. Yurke opens his tweezers through the exogenous addition of a displacement strand, but I suggest that we look for a way to bind some sort of ss DNA to the surface of a cell or cellular protein, so that when a nanobox approaches, the strand would unhook the latch.

Given the (hypothesized) large amount of cellular protrusions from E. coli, this could be difficult to engineer, and there may be a kinetic difficulty with the constraining of the displacement strand.

Yurke carried out the reaction in at 20°C SPSC buffer, which contains 50mM sodium biphosphate, 1M NaCl, and pH 6.5. We should investigate if strand displacement will work at physiological pH and lower salt concentrations as well.

Bibliography

1. Shih WM, Quispe JD, and Joyce GF. A 1.7-kilobase single-stranded DNA that folds into a nanoscale octahedron. Nature. 2004 Feb 12;427(6975):618-21. DOI:10.1038/nature02307 |

• Harvard link

2. Shih WM and Spudich JA. The myosin relay helix to converter interface remains intact throughout the actomyosin ATPase cycle. J Biol Chem. 2001 Jun 1;276(22):19491-4. DOI:10.1074/jbc.M010887200 |

• Harvard link

3. Shih WM, Gryczynski Z, Lakowicz JR, and Spudich JA. A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin. Cell. 2000 Sep 1;102(5):683-94. DOI:10.1016/s0092-8674(00)00090-8 |

• Harvard link

4. Yurke B, Turberfield AJ, Mills AP Jr, Simmel FC, and Neumann JL. A DNA-fuelled molecular machine made of DNA. Nature. 2000 Aug 10;406(6796):605-8. DOI:10.1038/35020524 |

• Harvard link

Other projects

My work in Biophysics 101

Contact info

Email me: (my last name) at fas dot harvard dot edu