After preparing the solutions now you are ready to produce small, medium or large scale cultures
The mutation in T. butanivorans G113N causes the cell to predominantly produce 2-butanol rather than 1-butanol when oxidizing butane. As a result this mutant strain requires two more steps to culture than for the wild-type strain
a. A cooling trap in a culture hood, i.e. set a refrigeration unit to 4oC with a glass trap (figure below).
b. A 125 L plastic carboy (figure below) with fresh air head space (e.g. blow fresh air with a fan to flush out spent head space from a previous culture)
c. Set up a GC with a TCD to measure butane concentration. GC conditions: carrier N2; column- Porapak T at 150 oC; Injector at 220 oC; Attenuation 32; Current 70 mA. Recorder conditions: 1 mV input and chart speed 30 cm/hr
Preparing the culture glass carboy (figure below):a.
To the sterile carboy containing 14.25 L of 1x salts, add the
following: 15 mL of vitamin solutions (sterile filtered), 7.5 mL of sterile 200
mM Fe3+-EDTA; 750 mL of sterile 20x phosphate buffer and 1 L of
confluent T. butanivorans cells (medium scale
culture)
b. Carefully place sterile aerating assemblage into the culture (figure below). Use a tape around the stopper to secure the assemblage and ensure no gas will leak
c. Wrap around a heating coil to the outside of the carboy
d. Bring the 15 L carboy into the hood, and place on stirring plate (figure below). Set the stirring to low. If one stirs fast the magnetic bar will tend to go to the side of the carboy and stop mixing, thus limiting gas exchange.
e. Fill temperature gauge tube in the bubbler stopper with water to ensure that the temperature regulator registers the correct temperature. Place temperature sensor in the tube. Place a thermometer to check that the temperature settings are correct during the incubation.
f. Plug heating coil into a temperature controller set to 30 oC. Turn on the power limited with a power controller set to ~45 Volts. Turn on temperature controller to start heating. One should feel heat from the coil on the carboy. Connect gas line from the air pump to the inlet gas line of the bubbler.
g. Connect the gas outlet line from the culture to the line that leads to the cold trap. Ensure the line from the cold trap to the large plastic carboy is connected. Ensure the line from the plastic carboy to the air pump is connected.
h. Turn on air pump. One should see bubbling in the culture glass carboy.
Preparing the 120 L plastic carboy with butane.a. Attach butane tank hose to the gas inlet line on the top of the carboy.
b. Turn tank on, let butane flow freely into carboy and stop after ~5 min.
Checking the concentration of butane in the carboy by GC.a. Sample 5 mL of the headspace with a 10 mL plastic syringe. Detach the syringe from the needle in the sampling port of the carboy and plug with a septum to prevent sample loss while transporting to the GC. Remove 100 µL of the gas in the syringe by piercing septum with Hamilton syringe and inject into GC. The first two peaks will be O2 and CO2. The third peak will be butane. THe concentration of butane should be 7 to 10% (i.e. the butane peak height should be about 4 cm high. Adjust the concentration in the carboy adding more or venting excess butane
b. Check thoroughly all connecting lines, inlet and outlet ports, stoppers, carboy seal and temperature regulator for leaks.
c. Culture should be confluent (OD600 ~1) in 24 hrs
Growing T. butanivorans G113N mutant in a large scale carboy:Everything is the same for the G113N strain except for the following:
a. Use 2 L of culture to inoculate the carboy instead of 1 L
b. Add 50 µM MnCl2 to the culture (7.5 mL of 100 mM MnCl2 stock, sterile filtered)
c. Add 10 µM Na2CO3 (0.75 µL of 200 mM Na2CO3 stock, sterile filtered, or in an autoclave)
