Order of Experiment:
- Obtain the mCherry gene and mutation target sequence via PCR.
- Clone the mutated region and mCherry into a PMEV4 vector.
- Clone the recombinant plasmid into E. coli to produce large amounts of plasmid.
- Transform the vector into M. Maripaludis WT and plate into anaerobic agar bottles.
- Inoculate isolated colonies into formate media.
- Aliquot and freeze some of each mutant for storage.
- Lyse the cells and extract the plasmids with mCherry.
- Measure fluorescence on the plate reader.
- Make Parts.
- Combine the following (make four replicates):
- ddH2O: 15.25uL
- Buffer mix: 5.75uL
- Primer mix: 2uL
- Template (MCherry Plasmid Control): 2uL
- Place sample in the PCR machine and run the iGEM Phusion Procedure. For amplifying pSB1C3, use pBrick-F/R; For amplifying inserts, use B-Fp/B-R.
- Mixture :
- ddH2O :
- 10x T4 DNA Ligase Buffer :
- T4 DNA Ligase :
- Insert :
- Plasmid :
- 20 µL
- 8 µL
- 2 µL
- 1 µL
- 2 µL
- 7 µL
Transformation via PEG (1.5mL method - Plating and Enrichment)
(As of May 19, 2015)
Check list of items on Day 1
- Prepare Minispin and Vortex
- Preheat sandbox
- TB and TB+PEG Buffer
- Recipient strain of OD 0.7-1.0 in a rack (always make at least 2 days ahead)
- Selection markers
- Media and plates
- Latex gloves, tube locks, tips, pipette (1mL), microcentrifuge tubes
- Plasmid concentration determined (0.4-0.6ug per sample)
- 100mL beaker (for discarding supernatant)
Day 1 (making plates)
- Melt plates (2 plates per sample) by autoclave, let cool down to 65C
- Add Na2S, antibiotics, and additives into plates and let solidify on side way
- Centrifuge at 3,900rpm (Minispin) for 10 minutes
- Discard supernatant, add 75uL of TB, pipetting up and down for 10 times
- Add 0.2-0.3ug of plasmid and 45 uL of TB-PEG, pipetting up and down for 10 times
- Incubate at 37C in sand box for 1 hour
- While waiting, prepare 5mL of broth media plus Na2S in balch tube
- After incubation, combine 2 micro-tubes of cells into 1mL broth (total vol=1.24mL), vortex (strong)
- Take 2 new micro-tubes and add 0.9mL broth to each, now you have -2mL broth left in balch tube
- Do 10^0, 10^-1, and 10^-2 serial dilution with cells from the previous step, vortex (strong)
- Plate 0.6mL of cells from 10^0, and plate 1mL from 10^-1 and 10^-2. Lay plates sideways overnight before incubating at 37C
- Inoculate remaining cells (~0.6mL) from 10^0 to the balch tube with 2mL broth, record OD and incubate overnight at 37C
Day 3 (enrichment in broth)
- Record OD again from the final step in Day 2, inoculate 0.5mL into 2 5mL of selective broth.
- Make frozen stocks of remaining.
- Determine which colonies to be inoculation from frozen stock and record on the google doc.
- Check chamber for syringes, media, needles.
- QUICKLY place selected cryotubes into the chamber from the freezer.
- Label 5mL media tubes with the Library number, colony number, date, and triplicate ID (if applicable).
- Add .05mL 100X pur and 0.1mL NaS to each needed media tube (2 per cryotube)
- Then vortex your crytotube and distribute the contents evenly (~0.3mL each) between the 2 prepared media tubes.
- Determine which sequences warrant sequencing. The sequences that are sequenced need to have a strong Band around 900BP and a level of fluorescence greater than that of the negative.
- Give the colonies new labels such as 1-10 that correspond with the a specific colony, For example L1C1A will be 1, L1C2A will be 2 and L1C1B will be 3.
- Spin PCR Product down
- Get 20ul and 200uL micro pipettes and tips
- Retrieve the DNA Clean&Concentrator-25(make sure it is 25 by checking size of columns)
- In a 1.5mL microcentrifuge tube, add 5 volumes of DNA binding buffer to 1 volume PCR Product
- Transfer Mixture to a provided Zymo-Spin Column in a collection tube
- Centrifuge for 30 seconds at 17g Discard Flow-through (set centrifuge timer to 1 minute and end 30 seconds early)
- Add 200 uL DNA wash buffer to the column, Centrifuge for 30 seconds at 17G
- Repeat previous step
- Add 25 uL water directly to column matrix and incubate at room temperature at room temperature for 1 minute,
- Transfer column to 1.5 ml microcentrifuge tube and centrifuge for 30 seconds to elute the DNA
- Remove Column
Making anaerobic formate media for Methanococcus maripaludis (400mL)
- Glass-distilled water: 120mL
- Glycylglycine buffer, 1M, pH=8.0: 80mL
- General Salts Solution: 200mL
- K2HPO4 14g/L: 4.0mL
- Na acetate-3H2O, 136g/L: 4.0mL
- Trace Mineral Solution: 4.0mL
- Iron Stock Solution: 2.0mL
- Sodium formate (NaCOOH): 10.8g
- Sodium Bicarbonate (NaHCO3): 1.5g
- Casamino acids: 0.8g
- Combine media ingredients and sparge with a stream of N2 gas for >60minutes
- Add 0.05g cysteine-HCl per 100mL and continue sparging for >10minutes.
- Place in the anaerobic chamber overnight
- Dispense media and pressurize to 15psi with N2/CO2 gas
Preparation of Sodium Sulfide
- Add 200mL of diH2O to flask and mark water line. Add 2 pellets of NaOH and 20mL more of ddH2O.
- Boil the 220mL diH2O while flushing flask with N2 until the water level reaches the original 200mL water line.
- Let flask cool while flushing with N2, move the flask to the gassing station in the fume hood. Continue flashing with N2.
- While flask is cooling, weigh our slightly moe than 5g Na2S-9H2O. For the following steps, work in the fume hood and wear gloves. Clean the sodium sulfide crystal by briefly rinsing the crystal with diH2O. To do this, swirl the crystal in a small beaker with the water. Dry the crystal by blotting with a paper towel or kimwipe. Reweigh the crytal to insure that the final weight is 90-110% of the desired weight.
- Add the clean, weighted sodium sulfide to the cooled flask while flushing with N2 and mix until partially dissolved.
- Stopper the flask, discontinue flushing, and transfer to the anaerobic chamber
- Dispense into tubes that have been equilibrated in the chamber for at least for one day to allow the O2 to desorb from the glassware. Use the thick butyl stoppers that are ungreased and have been equilibrated in the chamber for one day.
- Remove tubes from the chamber and pressure with N2 at 15 psi
- Autoclave on gravity cycle (rapid dry) for 20 minutes.
- Store in anaerobic chamber.
Making anaerobic agar plates
- Add 0.15g agar to each glass plate bottle and place in chamber for at least overnight.
- Make the full volume of media needed and also place in chamber overnight.
- Distribute 10mL media to each of the glass plate bottles.
- Stopper and seal the bottles.
- Pressurize each bottle to 15psi with N2/CO2
- Autoclave on gravity cycle with a 30 minutes sterilization.
- Remove from autoclave.