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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.
Restriction Digest
We set up our restriction digestions as suggested by New England Biolabs. We incubated the reactions at 37°C for 30 minutes and then heat inactivated for 20 minutes at 80°C.
OmpC Digestion:
25 ul Purified Plasmid (500 ng)
1 ul SpeI Restriction Enzyme
1 ul PstI Restriction Enzyme
5 ul 10X NEB Buffer 2
.5 ul 100X BSA
17.5 ul H20
Total: 50 ul
Banana Smell Generator Digestion:
11.4 ul Purified Plasmid (500 ng)
1 ul Xbal Restriction Enzyme
1 ul PstI Restriction Enzyme
5 ul 10X NEB Buffer 3
.5 ul 100X BSA
31.1 ul H20
Total: 50 ul
Gel Extraction/Fragment Purification
We used the Sigma GenElute™ Gel DNA Extraction Kit to elute the digested DNA out of the gel. Procedure A. Spin Procedure for Agarose Gels All centrifugations (spins) are performed at 12,000 to 16,000 x g (See Appendix I to convert g-force to rpm).
1a. Excise band. Excise the DNA fragment of interest from the agarose gel with a clean, sharp scalpel or razor blade. Trim away excess gel to minimize the amount of agarose.
2a. Weigh gel. Weigh the gel slice in a tared colorless tube.
3a. Solubilize gel. Add 3 gel volumes of the Gel Solubilization Solution to the gel slice. In other words, for every 100 mg of agarose gel, add 300 L of Gel Solubilization Solution. Incubate the gel mixture at 50-60 °C for 10 minutes, or until the gel slice is completely dissolved. Vortex briefly every 2-3 minutes during incubation to help dissolve the gel. Note: To adequately dissolve a gel with an agarose concentration greater than 2%, it is necessary to increase the ratio of the Gel Solubilization Solution volume to the gel weight to 6:1.
4a. Prepare binding column. Preparation of the binding column can be completed while the agarose is being solubilized in step 3a. Place the GenElute Binding Column G into one of the provided 2 ml collection tubes. Add 500 L of the Column Preparation Solution to each binding column. Centrifuge for 1 minute. Discard flowthrough liquid.
5a. Check the color of the mixture. Once the gel slice is completely dissolved (step 3a) make sure the color of the mixture is yellow (similar to fresh Gel Solubilization Solution with no gel slice) prior to proceeding to the following step. If the color of the mixture is red, add 10 L of the 3 M Sodium Acetate Buffer, pH 5.2, and mix. The color should now be yellow. If not, add the 3 M Sodium Acetate Buffer, pH 5.2, in 10 L increments until the mixture is yellow.
6a. Add isopropanol. Add 1 gel volume of 100% isopropanol and mix until homogenous. For a gel with an agarose concentration greater than 2%, use 2 gel volumes of 100% isopropanol.
7a. Bind DNA. Load the solubilized gel solution mixture from step 6a into the binding column that is assembled in a 2 ml collection tube. It is normal to see an occasional color change from yellow to red once the sample is applied to the binding column. If the volume of the gel mixture is >700 L, load the sample onto the column in 700 L portions. Centrifuge for 1 minute after loading the column each time. Discard the flowthrough liquid. Note: Do not be alarmed if the flow-through has changed color.
8a. Wash column. Add 700 L of Wash Solution (diluted from Wash Solution Concentrate G as described under Preparation Instructions) to the binding column. Centrifuge for 1 minute. Remove the binding column from the collection tube and discard the flow-through liquid. Place the binding column back into the collection tube and centrifuge again for 1 minute without any additional wash solution to remove excess ethanol. Residual Wash Solution will not be completely removed unless the flow-through is discarded before the final centrifugation.
9a. Elute DNA. Transfer the binding column to a fresh collection tube. Add 50 L of Elution Solution to the center of the membrane and incubate for 1 minute. Centrifuge for 1 minute. For efficient recovery of intact plasmid DNA, preheat the elution solution to 65 °C prior to adding it to the membrane. Eluting at 65 °C improves plasmid DNA recoveries by 2 to 3-fold. Yields of large linear DNA fragments (>3 Kb) can also be increased by up to 20% by preheating the elution solution to 65 °C.
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 a colloid 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 enzyme 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 phosphorylated 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 advice on why just that part is failing. We also transformed another promoter that we will use.
5/24/12
We communicated with a mentor from Ginkgo BioWorks, Alyssa Henning. She gave us multiple suggestions on how to improve the wiki, as well as giving us advice on how to move forward on our wet lab work.
6/11/12
Members of the group that are going to the conference met and determined a schedule for completing as much lab work as possible before the wiki freeze.
6/13/12
Made liquid cultures of the transformed bacteria containing the OmpC promotor, bacteria containing the mint smell generator, and bacteria containing the banana smell generator.
6/15/2012
Purified plasmids from all three liquid cultures, then used a spectrophotometer to quantify the DNA. Did restriction digests on OmpC promotor and the banana smell generator in preperation for ligation. Ran digests on gel to seperate out unecessary parts.
6/16/2012
Purified fragments from restriction digest gel in preperation for ligation. Made final changes to wiki.
Lab work to be done before Jamboree:
Calculate transformation efficiency of parts Ligate mint generator and OmpC promotor Run ligation gel Transform bacteria with ligated plasmid Smell!
