pORTMAGE
PROTOCOL DUMP PLEASE FORMAT
Multiplex Accelerated Genome Engineering (MAGE) using the pORTMAGE plasmids
Make a stock of MAGE oligos with total concentration of 3-5uM in water.
Transform target strain with a pORTMAGE plasmid.
Grow pORTMAGE-added strain in a culture tube for overnight in 30-32°C shaker with appropriate antibiotic (in LB miller or LB lennox).
Measure the overnight OD and dilute the culture to OD=0.05 in 5 ml LB lennox or SOC. Remaining cells can be cryostocked as a checkpoint culture.
Wait 2-3 hr for the cells to read the OD≈0.55-0.7. The cells grow slightly faster in SOC.
While the cells grow, set the water bath to 42°C, and chill microcentrifuge tubes, electorporation cuvettes and sterile distilled water in 4°C fridge. If centrifuge can be set to 0°C, do so, otherwise, chill rotor in -20°C freezer
Put the culture tube in shaking water bath at 42°C for 15 min and no more than 20 min.
Add 5 ml media (LB lennox or SOC) to a culture tube and put it in shaker (37°C) to warm up for at least 30 min. This tube will be used at step 16.
Chill tube after induction in ice bath for at least 10 min.
Take 1 ml from the culture tube, transfer it to a chilled microcentrifuge tube, centrifuge it at 13,200 RPM for 30 seconds in cold centrifuge, and dump the supernatant.
Wash the sample with pre-chilled water (add 1 ml water to the tube,pipette mix on ice, centrifuge, and dump the water)
Repeat step 11 once if the growth media is LB lennox and repeat step 11 twice if the media is SOC.
Add 45-50 ul of the MAGE oligos to the microcentrifuge tube, resuspending the cells and transfer them to an electroporation cuvette.
Incubate on ice 1 min and then put the cuvette into electroporator (Eppendorf 2510), set the voltage to 1800kV and electroporate.
Resuspend in 500 uL SOC, transfer to culture tube, and incubate 30-60 min in 30-32°C incubator.
Add antibiotic to prewarmed 5 mL LB/SOC from step 8 to maintain pORTMAGE plasmid. Then, transfer to recovering MAGE cells.
Put the tube into 30-32°C shaker to let the cells grow. This is the end of one cycle.
To perform more MAGE cycles, use the culture tube from step 17 and go to step 5. If stopping for the day, resuspend recovery cells in LB + pORTMAGE antibiotic instead of SOC and grow overnight. This cultures is then used to resume at step 4. If stopping for longer, cryostock O/N culture the following morning to store a checkpoint of the cells.
Curing pORTMAGE plasmids after integrating
Plate integration recovery broth on plate that is non-selective for your pORTMAGE plasmid at 37 C overnight. If the integration is expected to be lower efficiency, grow at 30 C overnight instead.
Pick 2-5 colonies of interest from plate and inoculate two tubes of LB (or SOC) per colony. Be sure to label tubes according to their antibiotics and associated colonies.
a. First tube contains selective media for your integrated construct of interest (if applicable).
b. Second tube contains media with the antibiotic associated with your pORTMAGE plasmid.
Grow cells 8-12 hours at 37 C. A colony that shows no growth in the pORTMAGE-selective media, while growing in the non-selective tube, is cured and can be used for cryostocks or other downstream applications.
If there is growth in both tubes, spin down the non selective tubes in centrifuge and streak out cells from pellet on a plate that non-selective for your pORTMAGE plasmid and incubate at 37 C overnight.
Repeat growth cycles at 37C as described above until the plasmid is cured.
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