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| (Based on [http://www.citeulike.org/user/madhadron/article/983616 Udou et al, 1982])
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| # Grow culture of smeg overnight
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| # Add 20% glycine to final concentration 1.2%, and incubate for 16-20h.
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| # Centrifuge at 1,200g for 15 minutes, decant supernatant. Resuspend in same volume SMM.
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| # Centrifuge again. Resuspend in same volume of P medium.
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| # Incubate for 16 to 20h
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| # Centrifuge at 300g for 7 minutes and discard pellet.
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| # Centrifuge supernatant at 1,200g for 15 minutes.
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| # Wash twice with SMM
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|
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| ===P medium===
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| (Additives per L of water)
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| * 120g sucrose
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| * 10g glucose
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| * 12g L-glycine
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| * 1g (NH_4)_2 SO_4
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| * 0.25g K_2 SO_4
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| * 2.03g MgCl_2 times 6H_2)
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| * 3.68 CaCl_2 * 2H_2O
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| * 0.1g KH_2 PO_4
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| * 0.025M [N-tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid (TES; pH 7.2)
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| * 50mg lysozyme
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| * 30mg lytic enzyme no.2
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|
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| ===SMM===
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| 0.5M sucrose + 20mM MgCl_2 + 0.02M maleate (pH 6.5)
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| ===Alternate Protocol===
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| (based on [http://www.citeulike.org/user/madhadron/article/1080971 Thacore et al, 1963])
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| *Spheroplasting agents: 100μg/mL lysozyme + 6.84mM EDTA
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| *Supportive agents: 0.34M sucrose, 1.1μg/mL Mg$^{2+}$
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| Just add to 7H9.
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| ===Notes===
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| Need to measure mycolic acid concentration to make sure that they really have sloughed off cell wall (Mike Glickman is the local expert).
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| Make spheroplasts, then try centrifuging them, plating them, etc. How rough can I be, and how much does it matter? Can spheroplasted smeg grow? In the presence of lysozyme + EDTA? What is it's growth rate?
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|
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| ===To Characterize Spheroplasts===
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| Measure change in peptidoglycan concentration upon spheroplasty.
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| '''GAG medium''': 7H9 + 0.02% Tween-80 + 1mM GlcNAc + 5mM glucose
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|
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| * Grow culture overnight in osmotically supportive medium, with or without lysozyme + EDTA
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| ===Experiments===
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| ====Spheroplast cells, check shape and viability====
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| '''Materials'''
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| * 200 plates LB agar + osmotic support
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| * 24 slides
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| * 28 cuvettes
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| * PBS
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| * 5x 7H9
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| * 0.5M sterile EDTA
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| * 20% dextrose
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| * 20mg/mL lysozyme
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| * 0.83 (10%) MgSO<sub>4</sub> in water
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| '''Media''': 5mL 5x 7H9 + 340μL 0.5M EDTA + 125μL 20mg/mL lysozyme + 7mL 20% dextrose + 275μL 0.83M MgSO<sub>4</sub> + 12.26 autoclaved water
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|
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| '''Directions''':
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| # Grow ''M. smegmatis'' overnight in 7H9.
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| # In the morning, wash the cells and inoculate new cultures to OD$_600$=0.1: 7H9, 7H9 + spheroplasting agents, 7H9 + osmotic support, 7H9 + spheroplasting agents + osmotic support. Make blank tubes for each of these media for use in the spec. Measure OD and plate, with and without centrifuging. Put on to shake.
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| # Measure OD and plate every 2h for 10h, again with and without centrifuging. Heat fix samples on labeled slides for later observation.
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