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Team:BVCAPS Research KS/Notebook
From 2012hs.igem.org
Contents |
Protocols
Transformation
We used New England Biolab's High Efficiency Trasformation protocol.
1. Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
Plasmid Purification
We used the Aurum Plasmid Mini Kit, with the spin method, for plasmid isolation and purification.
1. Transfer up to 12 OD•ml of plasmid-containing bacterial host to a 1.5–2.0 ml capped microcentrifuge tube (not provided). Pellet the cells by centrifugation for 1 min. Remove all supernatant by decanting or pipetting.
2. Add 250 μl of resuspension solution and vortex or pipet up and down until the cell pellet is completely resuspended.
3. Add 250 μl of lysis solution and mix by inverting the capped tube briskly 6–8 times. DO NOT VORTEX OR SHAKE. The solution should become viscous and slightly clear. Note: The neutralization solution should be added within 5 min after lysis.
4. Add 350 μl of neutralization solution and mix by inverting the capped tube briskly 6–8 times. DO NOT VORTEX OR SHAKE. A visible precipitate should form.
5. Centrifuge the neutralized lysate for 5 min. A compact white debris pellet will form along the side or at the bottom of the tube. The supernatant or cleared lysate contains the plasmid DNA.
6. While centrifuging the lysate, insert a plasmid mini column into a 2 ml capless wash tube (provided).
7. By decanting or pipetting, transfer the cleared lysate from step 5 to the plasmid mini column. Centrifuge for 1 min.
8. The wash solution is supplied as a 5x concentrate. Add 4 volumes (100 ml) of 95–100% ethanol or reagent-grade (denatured) ethanol before initial use.
9. Remove the plasmid mini column from the wash tube. Discard the filtrate from the tube, and replace the column into the same wash tube. Add 750 μl of wash solution and centrifuge for 1 min.
10. Discard the wash solution from the tube, and replace the column into the same wash tube. Centrifuge for 1 additional minute to remove residual wash solution.
11. Transfer the plasmid mini column to a 1.5–2.0 ml capped microcentrifuge tube (not provided). Add 50 μl of elution solution onto the membrane stack at the base of the column and allow 1 min for the solution to saturate the membranes. Centrifuge for 1 min to elute the plasmid.
12. Discard the mini column and store the eluted DNA at 4ºC.
Notebook
3/20/2012
Today we attempted to build a circuit and added our project description. We also tried to select parts to be used in our biological system, we selected a promoter that functions as coliroid and is named BBa_R0082
3/21/2012
We worked on selecting the parts we need and selected two mint producing parts we could attempt to use BBa_J45004 and BBa_J45099. The banana producing part is BBa_J45199, an enyzme generator that creates an enzyme to catalyze the process that causes isoamyl alcohol to break down into isoamyl acetate, which smells like bananas.
3/23/2012
Today we hit a magnificent breakthrough in wiki page editing. We can now call ourselves masters. We also planned the system we will create for our project.
3/26/2012 and 3/27/2012
Worked on creating a visual of our system. We are working on Tinker Cell to create the whole thing and have a diagram of what we are creating. We know that we will need the light sensor (Cph8) part name BBa_I15010 and the molecule activating our smells will be the OmpRp part name BBa_R0082
3/28/2012 - 4/3/2012
Tim did a whole bunch of research and modeling. Austin and Brandon double tasked with "Operation Superman" which was a ginormous success.
4/4/2012
Each of the plasmids needed were inserted into compatible bacteria and grown on plates. The bacteria with the light sensor gene failed to grow.
4/5/2012
Today we moved isolated colonies of each part to broth for growth. We also poured a bunch of plates!
4/6/2012
We worked on isolating the plasmids of each part.
4/9/2012
Today we set down with our instructor to describe the next steps in our project. We also worked on finding published papers of our parts to get a better understanding about them. We also learned that OmpRp is phosphated OmpR and is activated when there is no light. OmpR regulates our system so at night it will be activating the banana smell but we need a activator that will activate the mint during light.
4/10/2012
We set down and worked on modeling our system and came to a realization of what parts we need to add like OmpC and OmpF promoter.
4/11/2012
The systems we are creating were finalized on the Tinkercell program and uploaded to the wiki. We also collaborated with our mentors at Arizona State University. Tim began modeling on Jmol.
4/16/2012
We attempted another transformation on the light sensor, but were unsuccessful.
4/25/2012
New competent cells were delivered so we reattempted the transformation of the light sensor. The bacteria failed to grow on the kanamycin plates, but successfully grew on regular LB plates. Our longshot attempt to grow the bacteria on ampicillin plates also failed.
4/26/2012
We problem solved why our kanamycin plates continued to prohibit bacterial growth and set up the experiment to determine what aspect of the transformation created the failures.
4/27/2012
We attempted the transformation of a part unrelated to the project. The plasmid provides both ampicillin and kanamycin resistance so we will attempt to grow the bacteria on one plate with ampicillin and another with kanamycin. If it grows on both then the issue with our light sensor is in the part. If it only grows on the ampicillin then we have an issue with our kanamycin.
5/2/12
We tried to transformation our light sensor again and it failed so we tried to contact another mentor to get advise on why just that part is failing. We also transformed another promoter that we will use.
