| 120 L fermentor:
| Setting up and sterilize (afternoon
PLEASE READ: SAFETY UPDATE 27/2/2006
UNTIL FURTHER NOTICE:
Due to safety concerns, the fermentor
can only be used for pressure sterilisation with no one being
in the room while heat B or
cool A are on. You must follow this rule. If in any doubt,
ask Jan Löwe or Kate Michie (G6, 2969, 2200).
A very simple 120l E. coli fermentation in 2xTY:
for more details, please consult Olga's
- Switch on extract fan and aircon
- Switch on centrifuge, if not on
- Switch on steam (outside room, levers 1 and 2, slowly!)
- Empty condensate container (white plastic container behind
the fermentor, under the rack)
- Put in CEREX sensor (large exit hole up ) and connect (back
port, aligning the two red dots)
- Switch on CEREX controller. Press "Options" until
- Switch ON Fermentor (front panel: power switch 16 on and
- Agitator snd Heater switch ON
- Put in DO sensor (left front port).
- Calibrate pH sensor: connect the electrode to the orange-top
black cable and plug the thin green wire (earth) into the little
hole on the steal holder. On the side panel, press "Menu",
calibrate <enter>, sensor 2 <enter>, measurement
<enter>, buffer cal <enter>, auto <enter>.
Submerge the electrode into buffer 1 (pH 7) <enter>, wait
until stops flashing, "pH 7" <enter>. Submerge
the electrode into buffer 2 (pH 10) <enter>, wait until
stops flashing, "pH 10" <enter> , exit, rinse
the electrode and put into front port to the right.
- Make sure drain valve and sample port valve (green) are closed
(including the four adjacent steam valves)
- Fill vessel 1/3 way with demineralised water (hose pipe)
- Add 2xTY media powder (supplied by media kitchen) without
creating too much dust (shield to cover)
- Add 3 ml of Sigma 204 antifoam, wash the inner side of the
- Fill up to 120 L (liquid level just below the window)
- Add 100 ml 4N NaOH
- Wipe seal and close vessel lid (re-assemble exhaust, break
disk exhaust, foam probes: F goes into the left hole, dipped
to the upper rim of the lid, and HF goes into the right hole,
dipped ~ 6 cm). Tighten rings of foam probes, check that all
the clamps on the lid are closed. Press 'Alarm ack' (alarm acknowledge)
- Switch on all supplies (3 to 15). Press "Activate"
- Switch on stirrer to dissolve powder (Control: Ag(itation)
- Adjust pH as required with NaOH, 2xTY: pH 7.4 (set valve
TY-5N to ON, remove one foam needle, open top port)
- ALWAYS make sure that TY-5N is set back to AUTO once top
port is closed and foam needles are in place
- Set stirrer (Ag) to 100 rpm
- Check CEREX sensor is in "sterilise mode" (toggle
through menu with key 'Options')
- Sterilise (heat B 98°C, 20 min 121.5°C). This is
not OK for sugars. Press "Start"
- To sterilise open the steam valves (small black valves) next
to drain port and sample port
- BEFORE HEAT B IS REACHED (pressure
starting to rise) YOU MUST LEAVE THE ROOm AND NOT GO IN UNTIL
COOL B IS ON.
- Once cooling down, (cool A or cool B light is on) close these
- Wait until in growth mode (can be forced by setting growth
temp in sterilise to above the current temperature)
- Switch fermentor off by pressing "Deactivate"
- To leave O/N, switch off water (lever 14
| Fermentation (morning day 2)
- Press 'activate'. Machine should be in growth mode
- Switch water back on (lever 14)
- Activate temperature loop (36°C, AUTO)
- Stirrer 100-200, oxygen enrichment on (sp=0), air 60-100
(watch out for too much foam); NOTE: DO should be OFF
- Calibrate DO electrode (press MENU, until "cal sensor
1" <enter>; measurement <enter>; air cal
<enter>; 100% (or 750 mm Hg) <enter>; WAIT until
DONE appears, then, calibrate 0% oxygen: press MENU, until "cal
sensor 1" <enter>; measurement <enter>;
disconnect cable from exygen sensor at port, press zero <enter>;WAIT
until DONE appears, re-connect the cable to the oxygen probe
- Start CEREX: Switch CEREX back from "Sterilise mode"
(Cancel), then "Options" until "Quickstart"
appears <enter>; low level cal = 9(=yes);immerse in broth
<enter>; calibrate. <enter>;.Rvalue=6.6 <enter>;
cell mass profile, "Options"until OD profiles
choose user profile 0001 <enter>; new run? <enter>;
press 9=yes to verify the run; you will get the message "NEW
- Reduce air flow to 50
- To add antibiotic and to inocculate open valve TY-5N, remove
one foam probe, add through port (be careful with toothpick)
- make sure TY-5N is on AUTO again, Air flow back to 100
- Start BioCommand with recipe 'LMB standard recipe' and start
- Use Views, Trend.1 to monitor and print
- Samples can be taken through sample port (green) This requires
removing the lower steam connection.
- To add inducers etc., open valve TY-5N, remove foam probe
and add through port, set TY-5N to AUTO afterwards.
Assembling/disassembling the centrifuge
Footbearing: After taking the tubing out of the footbearing,
turn the levers upwards and remove the footbearing unit downward,
screw off the top lid and wash the spring within it and all the
parts, grease all the threadings, assemble
Grease the copper ring in the footbearing body, using the red
"syringe", turn the "syringe" knob 1/ 4 to
squirt the grease
Put the footbearing into the bottom of the centrifuge and push
Screw-in inlet tubing into the footbearing with the conical metal
spray sitting loosely on top of the inlet assembly
Cylinder bottom assembly It has 4 parts: large cylinder
bottom with spray holes, small propeller that fits into it, metal
disc with red rubber o-ring (do NOT grease it) and a large propeller.
Grease all the threads, but do not plug-up spray-holes at the
| Harvesting (evening day 2 or morning
- Stop all loops (not required, can be done at the end, in
this case reduce airflow to 50)
- Open valve TY-5N (=On), remove one foam sensor, open sample
port, close valve TY-5N again (=AUTO)
- Fermentor can be switched off at this point: "Deactivate"
and switch off all switches/levels No 17 to 1
- Attach pump (left tubing) to drain valve and open drain valve.
Right tubing to centrifuge.
- Assemble centrifuge (see above) and place cylinder (heavy,
DELICATE) using towel to protect the edge of the centrifuge drum
- Put on lid, take off plastic cap from top of cylinder, grease
- Push-in the head bearing spindle from the top, check that
teeth on the spindle alternate with notches
- Tighten (clockwise) with two spanners
- Lower large metal sleeve and tighten it
- Move kill tank under outlet, put spray tray under centrifuge
- Start centrifuge (noisy, use ear protection)
- When up to speed (15K) start culture pump at speed '10' (may
work at 20 as well), adjust so that eflux is clear (after debubbling)
- this takes roughly1 hour . Monitor frequently for leakage.
- Stop centrifuge. It takes 10 minutes before standstill light
comes on. It is a timer and does not detect rotation.
- Remove rotor, use protective cap on top of cylinder. Be careful
cylinder is VERY HEAVY and delicate (it's a rotor)!
- Put into brackets on rotor trolley, make sure draining tap
on cart is closed
- Remove lower srew cap with spanner
- Remove Teflon liner (tool), put onto area near sink
- Scrape cells together with shovel
- Package into many bags and flatten to enable fast
thawing, freeze in LN2
- Disassemble rotor completely and clean, including lower bearing
and inside of bowl.
- Clean trolley (attach hose to pump empty), rinse with water
- Leave rotor open to dry
- Clean all stainless steel parts of the fermentor, including
inside (hose pipe)
- Attach pump to drain valve to pump empty (several times)
- IF THE CLEANING TAKES YOU LESS THAN 1 HOUR, IT IS NOT
- It is mandatory to clean the floor with a mop at the very
end. Also make sure the sink is left tidy.
- Switch off fan and or aircon
| Biological containment
- We are required to contain all genetically modified organisms
and not to release them.
- The supernatant in the kill tank needs to be de-activated
using the protocol you have verified for your particular experiment
- If in doubt, ask Jo Butler and consult this
- After de-activation, the supernatant is pumped away and the
tank is cleaned with water.