Fermentation

Quad Parallel Fermentation

Day 1

SOLUTIONS /APPARATUS

  Acid line use 5% v/v 99.8 sulphuric/water)

  pH probe stored in 3M KCl

  use dilute ammonia (10 %)

  DOT probe must be connected 8 hrs pre-use – typically plug in O/N.

Method

Prepare 5 L defined media in 5L conical flask or equivalent

--pH from around 2 up to 6..95 with approx 25% ammonia. + acid [to 475 ml RO water, add 25ml 99% sulphuric acid carefully] + base [to 300 ml RO water add 200 ml 25% v/v ammonia stock, IN FUME HOOD] + Filt. Sterile antibiotic stocks

Autoclave 4 x l L conicals with 200 ml defined media in them. 4 x 1 L conicals with LB in them. 200 ml each. 1 x 2L of defined media into Durans (autoclave) pre-PPG addition 3 x Tubing for inoculum transfer. 1 x 100 ml Duran of defined media for OD measurement.

Book shakers.

Check Oxygen supplies!

Dry eppendorfs in Oven.

INOCULATION

SCHEDULE

Day 2

INOCULATION SCHEDULE

--use a scrape of glycerol stock to inoculate 5ml complex Luria Bertani broth, no antibiotic, grown in 25 ml universal.

--incubate at 30°C in an orbital shaker (200 rpm) overnight.

Day 3

INOCULATION SCHEDULE

--Use 5 ml LB culture to inoculate 200 ml of LB media, containing relevant amount of antibiotic for maintenance of plasmid(s).

Incubate at 30°C in an orbital shaker (200 rpm) for 4-6 hours to an OD600 1-3. Check OD600.

Weigh eppendorfs! Label fractionation eppendorfs.

Use 20 ml of over-day LB culture to inoculate 200 ml of defined media, containing relevant amount of antibiotic for maintenance of plasmid(s). Incubate at 30 C, 18-22 hours. 1.5-2.0

Midday

Initialise Fermenter apparatus:

  pH calibrate

  DOT check

-fill fermenter chambers with, typically 900ml media. Add PPG 0.2 ml/L.

--wool/foil all filters etc

--autoclave

prepare/calculate how much antibiotic/inducer is needed.

End of autoclave run –

--plug in DOT probes, pH probes

--connect acid / base lines, assess pumps

- connect condenser, etc.

Connect up laptop, etc.

Day 4

Confirm fermenter status – data logging etc, cascade control etc.

Measure OD600 of starter cultures.

INOCULATION /FERM.SCHEDULE

Use autoclaved tubing to suck out required volume of inoculum for fermentation

Acid line use 5% v/v 99.8 sulphuric/water)

pH probe stored in 3M KCl

use dilute ammonia (10 %)

DOT probe must be connected 8 hrs pre-use – typically plug in O/N.

5 L of defined media

ADJUST pH BEFORE AUTOCLAVING!!!

Chemical Mass Done? (NH4)2SO4 25 g NaH2PO4.H2O 14g KCl 19.38g MgSO4.7H2O 5g Trace elements 50ml Citric acid. H2O 20g Glycerol 150ml OR 450ml

ADJUST pH BEFORE AUTOCLAVING!!!