Team:Groningen/Growing Streptomyces

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Revision as of 17:04, 9 September 2010

-Make plates with a thick layer of medium, 100 ml medium per big Petri dish.Medium of Streptomyces is mannitol soya flour medium (MS or SFM) 300 ml per 1L bottle to avoid boiling over in autoclave

Agar 6 g
D-mannitol 6 g
Soya flour 6 g
Demiwater 300ml
Plating ΔrdeAB spores 10^5 /plate

-Incubate for 7 days at 30 °C

-Harvest mycelium with razorblade

Cell wall isolation

-Scrape the mycelium with a razor blade and resuspend in 15 mL demi-water.

-Run this suspension throught the cell disrupter at 13 kpsi 6X:

Clean all the parts off the cell distrupter with 70% ethanol, including the tower, make sure the nozzle is not blocked by running some ethanol through it.

Let the loading cilinder and the nozzle rest in ice on aluminium foil for 5/10 minutes to cool them off(prevents protease activity later on).

Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.

Press the loading cilinder on the tower tightl (don't be to careful)

Load no more than 15 mL of your filtered suspension

Put on the nozzle and the lit of the nozzle, tightly

The big cilinder can be placed on top of the nozzle/ loading cilinder, twist and turn to get it on and align the dent at the bottom of the cilinder with the dent on the french press

Adjust your pressure to 13 kpsi

Use the pin to start the machine, after two loud klblam!'s remove it

Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL

Run it through another 5X

Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice

Clean all parts of the french style cell distrupter with 70% ethanol

-Cook in 2% SDS (spin off your French press samples- 5000 rpm and 5 min- then add 2% SDS)

-Spin off, 4000 rpm, 5 minutes: Use pellet

-Cook in 2% SDS

-Spin off,4000 rpm, 5 min: Use pellet -Wash 10X with demi-water:

Resuspend
Spin off: Use pellet
Etc.

-Freeze pellet (For freeze drying: free it well)

Concentrations for antibiotics

Antibiotic Stock E.coli B.subtilis

Ampicillin (Amp) (in H2O) 50 mg/ml (4 °C) 100(μg/ml) (:500)
Kanamycin (Km) 10 mg/ml (4 °C) 20 (μg/ml) (:500) 10 (μg/ml) (:100)
Spectinomycin (Spec) (in H2O) 10 mg/ml (4 °C) 100(μg/ml) (:100) 100 (μg/ml) (:100)
Erythromycin (Ery) E.coli stock 10 mg/ml (-18 °C) 150 (μg/ml) (:66.6)
Erythromycin(Ery) B.subtilis stock 1 mg/ml (-18 °C) 2-5 (μg/ml)
Tetracyclin (Tet) (50% EtOH) 3 mg/ml (-18 °C) 12 (μg/ml) (:250) 6(μg/ml) (:500)
Chloramphenicol (Cm) (50% EtOH) 5 mg/ml (-18 °C) 10-15 (μg/ml) (:500/333) 5(μg/ml) (:1000)
Lyncomycin 12.5 mg/ml (4 °C) 12.5 (μg/ml) (1:1000)*
Hygromycin 50 mg/ml (4 °C) 125 (μg/ml) (:400)

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 -Freeze pellet (For freeze drying: free it well)
-'''Expression Chaplins in NZ8900'''
--Grow O/N culture in Ty with antibiotics (Km5 and Cm5)
--Measure OD600 (with 0.9 ml with 100 μL of O/N culture, number *10=OD)
--Inoculate 15ml TY (with ab) in 100 ml flask to OD=0.1
--Grow till OD600 is approx. 0.5
--Take 2 ml t=0 sample
--Induce with 1% subtiline
--Continue growing
--Take 2 ml samples every hour
-'''Chaplin purification'''
--5mg cell wall in 1.5 mL tube. Do not use gloves because static electric interaction
--Add app. 1 mL of TFA with a glass pasteur pipette.Do this in fumer and never leave TFA open for more than 30 sec!
--Vortex 1 min
--Spin down insoluble material for 10 min at 13000 rpm
--Transfer supernatant to new 1.5 mL eppendorf tube or 5 mL Greiner tube
--Dry TFA for 1-2 hours
--When tube is dry resolve in water or Tris buffer
-'''Sample treatment'''
--Take 2ml sample and spin down
-'''Supernatant'''
--1.5 ml ml of supernant in separate cup, rest of supernatant can be discarted (at this point you can store in freezer)
--Do TCA precipitation until drying pellet after washing with aceton
--Continue with TFA treatment
-'''Cell pellet'''
--TFA treatment
--Pellet of TFA treatment with cells should be airdried
--Lysed for half and out at 37 °C (P-buffer with 20 mg/ml lysozyme)
--Speedvacum
--TFA treatment
 '''Concentrations for antibiotics	'''