Protocol for E. coli Fermentation Last revised 4/25/2011

Protocol for E. coli Fermentation
Last revised 4/25/2011
Day 1
1. Unless starting from single-use glycerol stocks, streak plate with desired strain of cells and incubate
overnight.
2. Prepare pre-culture media and flasks. As a general rule, use 2xYTPG media for extract preparation
and 2xYT for anything else. See recipes below. You will need:
Two 5 mL culture tubes of LB or of other fermentation media.
Two 250 mL shake flasks containing 50 mL fermentation media each.
[Note: Autoclave the glucose solution separately and sterilely add it to the rest of the
concentrated media after autoclaving.]
Small container of blank fermentation media.
Recipe for 200 mL 2xYTPG media
Ingredient
Amount
(g)
Final Conc.
(g/L)
Concentrated media
Tryptone 3.2 16
Yeast extract 2.0 10
Sodium chloride 1.0 5
Potassium phosphate, dibasic (K 2 HPO 4 ) 1.4 7
Potassium phosphate, monobasic
.6 3
(KH 2 PO 4 )
Adjust pH to 7.2 using 5N KOH. Adjust volume to 150 mL using NP H 2 O.
Glucose solution
Glucose 3.6 18.0
Adjust volume to 50 mL using NP H 2 O.
Autoclave both solutions on L30 cycle. Combine to make 200 mL 2xYTPG
media.
Recipe for 200 mL 2xYT media
Ingredient
Amount
(g)
Final Conc.
(g/L)
Concentrated media
Tryptone 3.2 16
Yeast extract 2.0 10
Sodium chloride 1.0 5
Adjust pH to 7.2 using 5N KOH. Adjust volume to 200 mL using NP H 2 O.
Autoclave on L30 cycle.
3. Autoclave on Gravity 30 cycle:
Six 1 L centrifuge bottles and their caps
[Note: Autoclave the bottles upside-down to prevent warping.]
Cooling coils for harvesting, ends covered in foil
Spoon-shaped spatulas for scraping pellets from bottles

Day 2
4. Inoculate a 5 mL culture tube from a freshly streaked plate. Alternatively, inoculate from a single-use
glycerol stock thawed at room temperature. Grow culture for 7 hours at 37C on a shaker (225 rpm)
or rotator (speed 7). After 7 hours use this pre-culture to inoculate two 50 mL flasks of media, in a
pre-warmed 250 mL shake flask. Incubate overnight on a shaker (225 rpm) at 37C.
4. Prepare the tri-port assembly attached to three bottles. All tubing connections should be
zip-tied, and each piece of tubing should be clamped on both ends. Ensure that the triport
is in the closed position. For typical E. coli fermentations, bottles should contain
glucose feed solution (see recipe in Step 6), 5N potassium hydroxide (for pH control),
and water (to correct the working media volume for evaporative losses during
sterilization). One opening on each bottle cap should be connected to the tri-port by
tubing and this opening should be attached on the inside to tubing that runs to the bottom
of the bottle. The other opening should be attached to an air filter. The tubing to the air
filter should be clamped and the filter should be covered with foil to protect it from
moisture. Bottle caps should be loose during the autoclave run. Autoclave on Liquid 30
cycle, and write a tally on each of the air filters to record the number of autoclave runs
they have experienced.
[Note: Acids and bases should NOT be put in the autoclave. Instead, autoclave an
empty bottle and sterilely transfer acid/base into the bottle later.]
[Note: If acid feed is required, use Tygon rather than silicone tubing, as acid degrades
silicone rapidly.]
6. Prepare 10 L of 2xYTPG media. Make the glucose solution first, as this takes the longest.
Recipe for 10 L 2xYTPG media
Ingredient
Amount
(g)
Final Conc.
(g/L)
Concentrated media
Tryptone 160 16
Yeast extract 100 10
Sodium chloride 50 5
Potassium phosphate, dibasic (K 2 HPO 4 ) 70 7
Potassium phosphate, monobasic
30 3
(KH 2 PO 4 )
Adjust pH to 7.05 using 5N KOH. Adjust volume to 9.2 L using NP H 2 O. Sterilize
in place.
Glucose solution
Glucose 180.2 18.0
Adjust volume to .8 L using NP H 2 O. Prepare in 1L glass bottle that can be
autoclaved as part of the tri-port assembly (see Step 5).
Recipe for 10 L 2xYT media
Ingredient
Amount Final Conc.
(g)
(g/L)
Tryptone 160 16
Yeast extract 100 10
Sodium chloride 50 5
Adjust pH to 7.2 using 5N KOH. Adjust volume to 10 L using NP H 2 O.

7. Prepare wash buffer and store at 4°C. Below are recipes for two commonly used buffers:
Recipe for 1 L Buffer A (from ribosome prep)
Ingredient
Stock
Final
concentration concentration
Amount (mL)
Tris-HCl, pH 7.2 @4°C 1 M 20 mM 20
NH 4 Cl 3 M 100 mM 33.3
MgCl 2 1 M 10 mM 10
EDTA, pH 8.0 .5 M .5 mM 1
DTT 1 M 1 mM 1
Adjust volume to 1 L using NP H 2 O. Store at 4°C. Add DTT just before use.
Recipe for 1 L S30 Extract Buffer
Ingredient
Stock
Final
concentration concentration
Amount (mL)
Tris-OAc, pH 8.2 @
1 M 10 mM 10
RT
Mg(OAc) 2 1.4 M 14 mM 10
KOAc 6 M 60 mM 10
DTT 1 M 2 mM 2
Adjust volume to 1 L using NP H 2 O. Store at 4°C. Add DTT just before use.
8. Prepare the fermenter.
a. Turn the fermenter ON using the red switch on the side. Drain water out of the fermenter and
collect in the 5 gallon bucket. Ensure that the harvest port is CLOSED afterwards.
b. Remove the pH probe from its storage solution, calibrate, and insert into a port near the bottom of
the fermenter. This takes approximately 15 minutes. During setup and sterilization, ensure the pH
controller is OFF.
c. Remove pO2 probe and insert into another port near the bottom of the fermenter. This probe will
be calibrated after sterilization.
d. Fill the fermenter with media using a funnel through one of the ports on the top. Make sure that
the drain valve on the bottom of the vessel is closed before adding media.
[Note: Check to see if the pH probe reading approximately matches reading from tabletop pH meter.]
e. Make sure the two front ports on the top of the fermenter have good septa installed.
f. After autoclaving and sterilely adding acid/base, attach the trip-port assembly to the fermenter.
g. Sterilization checklist:
Open the valve labeled STEAM, located on the wall behind the fermenter. Pressure should
read 35-40 psi (Insufficient pressure will lead to long or failed sterilization cycles).
Open the air valve on the wall to the left of the fermenter. Adjust the regulator at the wall to
100 psi.

Check the air supply pressure valve, which is located on the lower left back corner of the
fermenter control unit, is about 2 bar.
Open the main water supply at the wall.
Check that the cooling water supply pressure is about 0.5 bar. The gauge is located directly
behind the stirrer motor.
Close the green valve on the exhaust condenser so that cooling water flow is OFF (this is
required to properly sterilize the entire filter).
Remove the light and plastic ring from the top of the fermenter to prevent melting during
sterilization.
On the air sparger attachment, switch to Ster setting (not Ferm).
Open the rotameter on the front of the control unit to about 10 SLPM. The Main control
screen can then be used to control the actual air flow. Trust the values on the screen, not on
the rotameter. Set the air flow to 4 SLPM.
h. On the Main control screen, press the Phases button. Choose StateStart. Sterilization takes
approximately 90 minutes.
i. Sterilize the sample and harvest ports. Remove the sleeves and open the green steam valves to
both ports. Liquid condensate should come through first. After steam begins to flow, close the
valves, return the sleeves, re-open the valves, and sterilize for 30 minutes.
j. When the sterilization cycle finishes, the control unit will beep and display a message. Press Enter
to acknowledge the message and silence the alarm. On the Main control screen, set the
temperature to 37°C.
[Note: Can skip to step o at any time.]
k. After the sterilization cycle is complete and the vessel has cooled, pump the glucose solution into
the fermenter. Unscrew the tri-port to the open position and remove tubing clamps. Install the
glucose feed tubing in the external red Watson-Marlow pump. Using the mechanical switch on
the pump box, set the pump in Auto mode (not Manual). On the Main control screen, choose
SUBAT. Adjust the Setpoint to 100%, and set the Mode to Auto. This pump is controlled as
Substrate A. Be sure to return later to turn the pump off.
l. At 37°C, adjust the pH to its working value of 7.1 (or adjusted value as determined from
comparison to benchtop pH readings). Install tubing into the BASE pump on the front of the
fermenter and turn on the pH controller. From the Main control screen, choose pH. Adjust the
Setpoint to 7.1 and set the Mode to Auto. Watch to make sure that base is flowing properly
through the tri-port assembly.
m. Under fermentation conditions, calibrate the pO2 probe. This takes about 20 minutes. Make sure
that:
Temperature = 37°C
Stir rate = 600 rpm
Air flow = 8 SLPM (Set using Main control screen, not the rotameter)
Air sparger is set to Ferm position
All readings are steady (takes about 10 minutes)
(i) Choose Calibration button. Under Sensors, choose pO2.
(ii) At the wall, close the air valve. Open the nitrogen valve.
(iii) Choose ModeCalib. Nitro. The program will wait for the system to reach steady
conditions before accepting a calibration value.
(iv) At the wall, close the nitrogen valve and open the air valve.
(v) A window will appear to begin the air calibration. Press ok.
[Note: Percentages refer to the ratio of air fed. 0% air means 100% nitrogen.]

(vi) Check the zero (0-15 nA) and slope (25-200 nA) for appropriate values.
n. If the fermenter is not going to be inoculated right away:
Turn the stirrer control off.
Turn the pH control off (BEFORE reducing temperature)
Set the temperature to 15°C.
Leave the air on 4 SLPM to maintain positive pressure.
Begin recording data on the PC. Monitoring the readings during down time will indicate
contamination or other problems.
(i) Open MFCS Shell using the desktop icon.
(ii) Choose RunOperator service.
(iii) Choose Start BatchStart Batch.
(iv) Choose ActionsSynchronize.
(v) Hit Plotting button and double-click on the current batch. Choose variables to
display. Useful values are CO2, O2, pO2, pH, stir rate, and temperature.
9. Prepare overnight cultures. Combine glucose with concentrated media solution. Inoculate two 50 mL
cultures from 5 mL starter culture tubes. Grow overnight for 14-hrs at 37 on a rotary shaker (280 rpm)
Day 3
10. Final preparations for fermenter:
Set the temperature to 37°C. Takes about 10 minutes to stabilize.
After the temperature has stabilized, turn the pH controller on. Set to pH 7.1 (or adjusted
value).
Turn the exhaust condenser water flow on using the green valve.
Calibrate the pO2 probe if not already done. (See step 6.n.)
On the Main control screen, set the air flow rate to 8 SLPM.
Set the stir rate to 600 rpm.
11. Inoculate the fermenter.
a. Measure the OD 600 of overnight cultures using 1:20 dilution in blank media. Calculate inoculum
volume below:
V starter *OD starter = V ferm *OD init
The OD init is calculated from the desired OD at harvest and the number of doublings i (6-8 is
acceptable)
OD init *2 i = OD harvest
b. Sterilely collect a sample of blank media.
(i) Remove sample port sleeve and open valve to blow out condensate. Return sleeve.
(ii) Sterilize the sample port once between each sample removal. Open green valve to
flow steam for 15 minutes. Close the valve.
(iiI) Use an autoclave glove to remove the port sleeve.
(iv) Open the lever valve to remove ~10-20 mL of media. This is waste.
(v) Collect a ~20 mL of media for OD measurement (blank) and dilutions.
(vi) Open the green valve to flow steam BEFORE replacing the sleeve. This step
washes any leftover media through the line.
(vii) Replace the sleeve and sterilize for 15 minutes.
c. Sterilize the septum injection port by removing the plug and filling it with 70% ethanol. Using a
sterile syringe and Gauge 20 needle, inject the desired amount of overnight culture through the
septum into the fermenter. To maintain sterility, load the syringe in the biosafety cabinet or next
to a flame (the butane torch works well).

d. Upon inoculation, synchronize the batch by right clicking on the batch in the Batch Management
indow list and choosing Synchronize.
e. Take the OD of a small sample immediately after inoculation (see Step 9.b). This reading should
match the calculated OD init .
12. Monitor the fermentation. Take OD measurements approximately every thirty minutes. Track the
doubling time as the fermentation proceeds. Doubling time is typically 5-10 minutes longer in the
fermenter than in shake flasks.
13. Prepare to harvest cells.
Be sure that the harvest port is sterile as the OD approaches its final value.
Cool Beckman Avanti high speed centrifuge to 4°C.
Place Beckman JLA 8.1 rotor in the dairy case. Do NOT cool inside the centrifuge.
Pre-weigh 50 mL falcon tubes.
Make sure the harvesting coils are sterile.
Cools Thermo Multifuge (benchtop centrifuge) to 4°C.
14. Harvest the cells at desired OD (typically OD 3-3.3).
a. To harvest, change fermenter settings below:
Set the air flow rate at 4 SLPM.
Set the stir rate to 100 rpm.
Turn off the pH control.
Set the temperature to 4°C.
b. Remove the sleeve on the harvest port. Attach the cooling coil to the port and drain culture into
sterile 1 L centrifuge bottles. Balance the bottles. After the first six bottles are full, continue
collecting culture in sterile 2 L bottles.
c. Spin bottles in the Beckman Optima high speed centrifuge at 6000g at 4°C for 15 minutes to
harvest. Some strains (such as KC6) require a longer spin.
d. Remove supernatant by pouring. Dispense the remaining culture in centrifuge bottles on top of
the first round of pellets. Re-centrifuge.
e. Once all the cells have been pelleted, use a spoon-shaped spatula to transfer cells to pre-weighed
50 mL Falcon tubes. Resuspend cells in 20-30 mL wash buffer by vortexing and pipetting up and
down. If desired, use VWR tissue homogenizer on the second-lowest setting.
[Note: Rinse homogenizer with 70% ethanol before and after use.]
f. Centrifuge Falcon tubes in the Thermo Multifuge (fixed angle rotor) at 5000g and 4°C for 15
minutes to pellet cells. Decant supernatant by pouring.
g. Wash cells again as in steps (e-f).
h. Drain pellets upside down in the dairy case for 10 minutes.
i. Determine mass of each cell pellet. Flash freeze on liquid nitrogen and store at -80°C.
Cleanup:
Note: If the fermenter is not going to be cleaned the same day as the fermenter run it can be just rinsed
well with water and left filled with some water until the next day for cleaning.

15. Turn all controllers off except the air. Leave the air flow on at 4 SLPM to keep the culture out of the
air sparger. Release the pressure in the fermenter by opening the back regulator. Drain the
fermenter and turn the air flow off.
16. Remove the pH and PO2 probes from the fermenter and rinse them with water. Store the pH
electrode in 3M KCl. Store the pO2 probe in pO2 storage solution. Insert stoppers in the ports for the
probes.
17. Disconnect the tri-port assembly. Use a syringe to empty the acid and base feed lines back into the
stock bottles, or alternatively, one can switch the feed lines on the pump the acid and base back into
the bottles. Use a disposable syringe to flush the lines with water after they are empty.
18. Disconnect all wires, tubing, plugs, and connections from the fermenter lid. Use the hex wrench to
remove the two screws holding the motor in place. Remove the motor and set it on its stand.
19. Disassemble the exhaust filter housing and inspect the filter for damage or excessive accumulation of
dirt. Unscrew the pressure sensor from the block at the top of the exhaust cooler and set it aside.
20. Unscrew the four large bolts that hold the lid on and lift the lid off. Be sure not to scratch the interior
of the fermenter. Lay the assembly on the rim and the handle with the impeller and sparger sticking
up in the air. Use the crescent wrench to loosen the sparge tube nut and disconnect it. Remove the
screws at the bottom of sparge ring.
21. Use squirt bottles of acetic acid and water to rinse the interior of the fermenter. To preserve the
polish finish, do not scrub.
22. Wash all parts of the fermenter (except o-rings and filters) as follows:
(i) Wash with liquinox solution.
(ii) Rinse with 5% (v/v) acetic acid. There is a wash bottle by the sink for this.
(iii) Rinse with DI water.
(iv) Reassemble the fermenter Make sure the screws are in the sparge ring ring and it is
attached to the lid before replacing the lid.
23. Replace all plumbing and electrical connections.
[Note: Each connection fits in only one location, EXCEPT the Foam and High Foam probe wires.
Assembly of foam probe.

Assembly of condenser unit.
24. Fill the fermenter with 6-8 L of DI water and sterilize if it will not be used in the next 2-3 days.
After sterilization, turn off the control unit and close the air, water, and steam supply valves. Turn off
flow at the wall first, and then turn them off at the fermenter.