1. PMID: 8407909
2. PMID: 16493417
MscL and MscS are mechanosensitive ion channels, respectively triggered due to large or small distending of their bacterial cell's lipid bilayers. 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 a
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 PRISE Summer 2006 Cell: 781 330 1969 chan.tiffany at gmail.com