Team:Groningen/Growing Streptomyces

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Latest revision as of 17:19, 9 September 2010

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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--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:
-  a.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.
-  b.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).
-  c.Filtrate your samples using a 10 mL surringe and the filter device, catch your filtrate in a clean greiner tube.
-  d.Press the loading cilinder on the tower tightl (don't be to careful)
-  e.Load '''no more than 15 mL''' of your '''filtered''' suspension
-  f.Put on the nozzle and the lit of the nozzle, tightly
-  g.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
-  h.Adjust your pressure to 13 kpsi
-  i.Use the pin to start the machine, after two loud klblam!'s remove it
-  j.Collect the distrupted cells( they are in the nozzle), using a surringe of 10 mL
-  k.Run it through another 5X
-  l.Collect your severely distrupted cells using the surringe, collect them in a greiner tube and put that on ice
-  m.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)
-'''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
-'''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	'''