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| | == Time-series Microscopy of Colicin Killing == | | == Time-series Microscopy of Colicin Killing == |
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| - | started on Friday, Oct 25, 2008
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| - | MICROSCOPE
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| - | delta-vision from Applied Precision
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| - | STRAINS
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| - | prey: contains plasmid for ahl producer (luxI) and constitutive GFP
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| - | killer: contains plasmid for mcherry (arabinose induced at 0.01% v/w) and colE1 gene, which is AI-1 (ahl) induced
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| - | MATERIAL
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| - | TB medium
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| - | large labtek chamber
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| - | PURPOSE
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| - | show killing effect of prey strains by killer cells in the vicinity of prey cells. killer cells far away from prey cell will not lyse due to missing ahl induction.
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| - | PREPARATION
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| - | Friday evening
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| - | TB 0.4% agar, 0.01% arabinose, top 1 ul of prey and killer stock, bottom 1ul of killer stock
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| - | [in addition: TB 0.2% agar, LB 0.2% agar, and LB0.4% agar]
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| - | inoculation of prey and killer strain in tb and lb medium without arabinose
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| - | Saturday morning:
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| - | dilution of prey and killer strain in tb medium with arabinose
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| - | starting new chambers from cultures without arabinose, mcherry not induced:
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| - | 1) Marking:
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| - | TB 0.4% agar, 0.01% arabinose
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| - | 1x) top 0.5 ul of prey and 0.5 ul of killer culture, bottom 0.5 ul of killer culture
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| - | 5x) top 0.5 ul of prey and 2.5 ul of killer culture, bottom 2.5 ul of killer culture
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| - | 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture
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| - | 2) TB without agar, 0.01% arabinose
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| - | 1x) top 0.5 ul of prey and 0.5 ul of killer culture, bottom 0.5 ul of killer culture
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| - | 5x) top 0.5 ul of prey and 2.5 ul of killer culture, bottom 2.5 ul of killer culture
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| - | 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture
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| - | chambers are incubated at room temperature
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| - | Saturday evening:
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| - | dilution of prey and killer strain in tb medium with arabinose
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| - | starting new chambers from cultures with arabinose, mcherry induced):
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| - | 1) TB 0.4% agar, 0.01% arabinose (Marking: 0.4 tb ara)
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| - | 1x) top 0.5 ul of prey and 0.5 ul of killer culture, bottom 0.5 ul of killer culture
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| - | 5x) top 0.5 ul of prey and 2.5 ul of killer culture, bottom 2.5 ul of killer culture
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| - | 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture
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| - | 2) TB without agar, 0.01% arabinose (Marking: 0.0 tb ara)
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| - | 1x) top 0.5 ul of prey and 0.5 ul of killer culture, bottom 0.5 ul of killer culture (culture with arabinose, mcherry induced)
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| - | 5x) top 0.5 ul of prey and 2.5 ul of killer culture, bottom 2.5 ul of killer culture (culture with arabinose, mcherry induced)
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| - | 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture (culture with arabinose, mcherry induced)
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| - | MEASUREMENTS
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| - | - measurement on saturday (measurement 14h): 20081025_kp_tb_14h_scan*
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| - | co-inoculated killer and prey cells lead to a dominance of prey cells, probably because prey cells are numerous enough to produce enough autoinducer that leads to self-killing of killer cells. Since killer cells do not become numerous, not enough ColE1 can be excreted to induce a toxic effect on the prey cells
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| - | - measurement after 20h (20081025_pk_20h_u* and 20081025_pk_20h_d*)
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| - | all killer cells have disappeared, prey cells are present in abundance. --> not true, mCHERRY was not completely expressed!!
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| - | - measurement on saturday night, sunday, and monday of cultures in TB with/without agar (1x, 5x, and 50x)
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| - | files saved with following pattern:
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| - | without agar:
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| - | 20081025_pk_ho_1x_2h_R3D.dv: date, experiment (prey/killer), homogeneous (without agar), 1:1 killer/prey ratio (5x=5:1 killer/prey ratio), after 2 hours and all other times, respectively.
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| - | with agar:
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| - | 20081025_pk_fi_1x_2h_R3D.dv: date, experiment (prey/killer), fixed (with agar), 1:1 killer/prey ratio, after 2 hours and all other times, respectively.
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