- MicroCon Assay
- 1 tube 6hb
- 1 tube c5.0
- must make a >15-lane 2% Mg2+ agarose gel
- PEG Precipitation of Mg2+, 6x Folding Reactions
- aliquot 20uL for a gel immediately
- must make a 7-lane gel
- with the other 20uL aliquot, PEG precipitate:
- add 20uL 20% PEG
- dilute (currently 5M) 20uL NaCl with equal volume 20uL H2O
- add 10uL (now 2.5M) NaCl
- aliquot 20uL for a gel immediately
- Organize and Wiki-Table the c5.0 Latches and Attachment Oligos
(* Microcon the Superconcentrated Biotinylated Samples from Saturday)
- do only if the 6hb, c5.0 works
- Folded 8 40uL rxns each of v4.0 Eb and Gb (protocol)
- Design Matt's oligos with the AscIII site
- Add to order form for v5.0 latches, send to Invitrogen
- Make Mg++ gel (add Mg++ after agarose added?), Mg++ buffer (protocol)
- Run 4 samples each of Gb and Eb
- Gel extract all 4 samples each using Qiagen, 50uL EB elution.
- Run 5uL on another Mg++ gel.
- PEG precipitate 4 samples each of Gb and Eb at 10+% (protocol)
- Run 5uL on Mg++ gel.
- Incubate with streptavidin (protocol).
- Run, in highly concentrated form (ie. 2 lanes, 1 sample of half of each purification for each lane), on two different PA gels.
- Stain one gel with Coomassie blue (protocol).
- Stain other with SYBR Gold (protocol).
- Pray; post pics.
Relevant Papers Summaries
- Paborsky LR, McCurdy SN, Griffin LC, Toole JJ, and Leung LL. The single-stranded DNA aptamer-binding site of human thrombin. J Biol Chem. 1993 Oct 5;268(28):20808-11.
Thrombin, a serine protease functioning in the blood clotting pathways, is bound by a DNA aptamer with the sequence GGTTGGTGTGGTTGG; this binding inactivates thrombin. Assays of the aptamer's binding site and specificity were done, showing that the aptamer binds thrombin over competitive analogues. Assays also showed that the aptamer binds thrombin subunit alpha-thrombin rather than gamma-thrombin, which is the active-site-containing part of thrombin - in other words, the aptamer doesn't change the functionality of the active site, but it does change the ability of the protein to bind its substrate in the necessary way for cleavage. The group pinpointed the binding activity to two specific lysine residues in the protein, Lys 21 and 65.
- Perozo E. Gating prokaryotic mechanosensitive channels. Nat Rev Mol Cell Biol. 2006 Feb;7(2):109-19. DOI:10.1038/nrm1833 |
MscL and MscS are mechanosensitive ion channels, named for their respective large or small conductances. Their mechanosensitivity to lateral distension across the lipid bilayer They are in effect a 'last ditch response' to physical stress which might result in fatal osmolarity changes. MscL is a five subunit protein, each subunit being comprised of two transmembrane helices; MscS is also a five subunit protein, each subunit comprised of transmembrane helices with a one cytosolic section. MscL has a 2-angstrom diameter pore when closed, but MscS has been resolved with an 11-angstrom pore - whether this is true when MscS is in open or closed state has still to be determined, due to water dynamics studies showing that in this 11-angstrom wide state, the hydrophobic residues may from a "vapor lock," preventing any solubilized ion from passing through.
1. RapidLigated 6uL of the insert (E0 fragments) and 1uL of the vector (R0 cut).
2. Transformed and plated the entire 21uL of ligation reaction onto Carb plates.
Goal: Digest and extract out the E0-gfp and ligate after the R0-promoter, into that pre-cut plasmid.
- R0: ---|E--|X---lacprom---|S--|P---
- E0: ---|E--|X-----gfp-----|S--|P---
1. Miniprepped the 3 BioBricks Plasmids (according to standard protocol)
- Made glycerol stocks of 1mL of the cultures from each transformant:
666.6uL of 50% glycerol (to give 20% glycerol total) 1mL of transformant culture
2. Double digested the 2 R0 (lac promoter) and 2 E0 (gfp) samples
- NB: Due to the NanoSpec reading out negative DNA concentrations, we proceeded with an arbitrary amount of transformed DNA for the digests.
- Digested with SpeI and PstI (NEBuffer 2) for R0, XbaI and PstI (NEBuffer 3) for E0, with the following protocol:
DIGEST PROTOCOL ---------------- 8uL miniprepped DNA 2.5uL 10x NEBuffer 2.5uL 10x (diluted 1:10 from the 100x stock) BSA 1uL enzyme 1 (diluted 1:2 on Parafilm from the stock, of which we would have needed 0.5uL) 1uL enzyme2 (1:2) 10uL dH2O ---------------- For a TOTAL VOLUME of 25 uL Incubated 1 hr @ 37%degC
- Deactivated the enzymes by placing on 80%deg heat block for 15 min.
3. Dephosphorylated 2 R0 samples to prevent self-ligation
- Added 1 unit (1uL of 1:10 dilution from the 10,000U/mL stock, of which we would have needed 0.1uL) of CIP (Calf Intestine Phosphatase) to the 2 R0 reactions.
- Incubated at 37%deg for 1hr.
4. Ran R0 and E0 samples on gel to separate out the gfp from the E0-plasmid
- Ran at 130V for ~45 min.
5. Gel extracted R0 plasmid (~2.1 kb, linearized by digestion) and E0 gfp fragment (~0.9kb)
- Cut out the 1 bright E0 0.9kb band and the 2 bright R0 2.2kb bands
- Extracted using the standard Qiagen protocol.
- Froze overnight at -20%deg.
1. Picked Colonies of Existing BioBrick Transformants
- Picked and numbered:
3 colonies of E7, 2 colonies of E0, 2 colonies of R0.
- Grew each colony in 5mL LB + 5uL of 50mg/mL Amp (1:100 dilution).
2. Ran Gel of DNA Nanostructures
- Ran 2% agarose gel of the nanostructure reaction and the 2 controls for ~40 min at 130V. (NB: Only left four lanes are ours.)
1kb Ladder | Oligo + Scaffold | -Oligo | -Scaffold
1. Folded DNA Nanostructures Using Shawn's Pre-designed Oligo Staples and Scaffold ssDNA
- Three reactions below were mixed according to given protocol.
oligos + scaffold -oligos -scaffold
2. Transformed Bacteria with Existing BioBrick Plasmids
- Transformed each BioBrick plasmid below into OneShot Top10 competent cells (Invitrogen) according to the following protocol.
R0010 (lac operon promoter), E7104 (T4 promoter + gfp), and E0241 (gfp).
Thaw cells; aliquot out ~30uL per transformation. Add desired amount of plasmid DNA (1 uL in this case?) to cells and leave on ice for 30 min. Heat shock at 37°C for 30 sec in a heating block. Ice for 2 min. Shake in 37%deg incubator for 1 hr. Pour onto plates with the required antibiotic (carbinomycin in this case); spread with beads or plate spreader. Incubate plates in 37%deg incubator overnight.
- Past iGEM Projects Presentation: UCSF
- Prospective Projects Research:
Lactobacillus Hijacking Receptor-based DNA Nanostructure Latch Biocryptography w/ DNA Nanostructures ?
Tiffany Chan Biochemical Sciences Kirkland '07 chan.tiffany at gmail.com