Neurosporene
PROTOCOL DUMP PLEASE FORMAT
Written by Chiam Yu; modified from Iman’s protocol
Note: Instruction in red color can be modified.
Start overnight culture of EcHW2f strain harboring pBad-crtEBI in25ml of LB(CM50) in a 125 ml-flask.
After around12-14 hoursculture at 250 rpm, 30°C, measure OD600. Then, resuspend cells in25ml of 2x M9 + 10 ug/ml CM with glucose 0.4%w/v + 0.2 mM of IPTG+ 10 mM Arabinose in a 250 ml-flask to an initial OD600 of 0.05. (If using ECNR2 derived strains, supplement with 0.25 ug/ml biotin)
Incubate at250 rpm, 37°C for exactly 10h.
Label microcentrifuge tubes according to the number of samples and weigh all them using the weighing machine with precision up to 4 decimal places. Also, change the oven temperature to 55°C.
Transfer 10 ml of the culture to 14ml-falcon tube using a motorized pipet. (Reuse the pipet to reduce the amount of waste).Pellet the cellsby centrifuge at max RPM of the tabletop centrifuge, RT for 5 minute.
Remove the supernatant and repeat step 4 to get more cells if the pellet is too small.
Resuspend the cell pellet in 1ml of sterile distilled water and transferred to a microcentrifuge tube (from step 4).
Centrifuge to spin down the cell at 15000 rpm, for 30s. Remove the supernatant using blue and red pipet.
Transfer about 50 ml of acetone to a new 50-ml centrifuge tube. Resuspend the cells in 1ml of acetone. (Use the motorized pipet with 1 ml sterile pipet. Acetone dissolves the pipet, so instead of measuring from 0 to 10 on the scale, measure from -1 to 9.)
DO NOT USE ACETONE NEAR ANY FLAME/FIRE. Perform the experiment in the hood.
Wrap the cap of the microcentrifuge tube with parafilm to prevent evaporation of acetone. Vortex the cell and acetone mixture.
Incubate the microcentrifuge tube in 55°C oven for 20 minutes. Vortex the tube every 5 minutes to ensure . (Acetone dissolve neurosporene best at 55°C)
Centrifuge the mixture for 5 min at 15000 rpm.
Meanwhile, prepare new microcentrifuge tube with 1ml of acetone each. (Use motorized pipettor).
For each sample, transfer 50 ul of supernatant from step 11 to the new tube with acetone in step 13.
Set nanodrop to UV-VIS mode, blank it with acetone using disposable (plastic) UV-cuvette. (VWR Catalog No: 47727-024 BRAND UV-Cuvette semi-micro is non-dissolvable in acetone)
Measure neurosporene at 470 nm.
Open the cap of the microcentrifuge tube containing the cells and remaining acetone, cover the whole rack with plastic wrap and air-dry the acetone in 65°C oven for 2 days.
Take out the microcentrifuge tube and close the cap tightly. Let the microcentrifuge tube equilibrate with room temperature for 3 - 4 hours, and then measure the weight of the tube plus dried cells. Back calculate the amount of cells in the tube.
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