UCL-Fermentation

From 2010.igem.org

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''ADJUST pH BEFORE AUTOCLAVING!!!''
''ADJUST pH BEFORE AUTOCLAVING!!!''
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! Antibiotic
 
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! Concentration
 
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| Ampicillin
 
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| 100 μg/mL
 
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| Bleocin
 
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| 5 μg/mL
 
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|-
 
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| Carbenicillin
 
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| 100 μg/mL
 
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|-
 
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| Chloramphenicol
 
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| 25 μg/mL
 
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|-
 
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| Coumermycin
 
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| 25 μg/mL
 
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|-
 
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| Gentamycin
 
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| 10 μg/mL
 
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| Kanamycin
 
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| 50 μg/mL
 
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|-
 
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| Spectinomycin
 
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| 50 μg/mL
 
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| Tetracycline
 
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| T7 10 μg/mL
 
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|}
 
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Example: To make 100 mL of LB/ampicillin growth media, add 100 μL of a 100 mg/mL ampicillin stock (1000X stock) to 100 mL of LB.
 
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===Reagent List: Antibiotics===
 
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Unless otherwise indicated, the antibiotic powder can be dissolved in H20. Addgene recommends making 1000X stock solutions and storing aliquots at -20oC.
 
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'''''Reagent Catalog Number'''''
 
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Revision as of 15:04, 30 September 2010

UCL IGEM 2010

RETURN TO IGEM 2010

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.


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!!!

Antibiotic Mass/Volume
(NH4)2SO4 25g
NaH2PO4.H2O 14g
KCL 19.38g
MgSO4.7H2O 5g
Trace elements 50 ml
Citric acid. H2O 20g
Glycerol 150ml/450ml

Chemical Mass Done?

ADJUST pH BEFORE AUTOCLAVING!!!

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