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Team:Heidelberg/Notebook/Visualization/Notebook/9thweek
From 2008.igem.org
Time-series Microscopy of Colicin Killing
started on Friday, Oct 25, 2008 MICROSCOPE delta-vision from Applied Precision STRAINS prey: contains plasmid for ahl producer (luxI) and constitutive GFP killer: contains plasmid for mcherry (arabinose induced at 0.01% v/w) and colE1 gene, which is AI-1 (ahl) induced MATERIAL TB medium large labtek chamber PURPOSE 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. PREPARATION Friday evening TB 0.4% agar, 0.01% arabinose, top 1 ul of prey and killer stock, bottom 1ul of killer stock [in addition: TB 0.2% agar, LB 0.2% agar, and LB0.4% agar] inoculation of prey and killer strain in tb and lb medium without arabinose Saturday morning: dilution of prey and killer strain in tb medium with arabinose starting new chambers from cultures without arabinose, mcherry not induced: 1) Marking: TB 0.4% agar, 0.01% arabinose 1x) top 0.5 ul of prey and 0.5 ul of killer culture, bottom 0.5 ul of killer culture 5x) top 0.5 ul of prey and 2.5 ul of killer culture, bottom 2.5 ul of killer culture 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture 2) TB without agar, 0.01% arabinose 1x) top 0.5 ul of prey and 0.5 ul of killer culture, bottom 0.5 ul of killer culture 5x) top 0.5 ul of prey and 2.5 ul of killer culture, bottom 2.5 ul of killer culture 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture chambers are incubated at room temperature Saturday evening: dilution of prey and killer strain in tb medium with arabinose starting new chambers from cultures with arabinose, mcherry induced): 1) TB 0.4% agar, 0.01% arabinose (Marking: 0.4 tb ara) 1x) top 0.5 ul of prey and 0.5 ul of killer culture, bottom 0.5 ul of killer culture 5x) top 0.5 ul of prey and 2.5 ul of killer culture, bottom 2.5 ul of killer culture 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture 2) TB without agar, 0.01% arabinose (Marking: 0.0 tb ara) 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) 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) 50x) top 0.5 ul of prey and 25 ul of killer culture, bottom 25 ul of killer culture (culture with arabinose, mcherry induced) MEASUREMENTS - measurement on saturday (measurement 14h): 20081025_kp_tb_14h_scan* 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 - measurement after 20h (20081025_pk_20h_u* and 20081025_pk_20h_d*) all killer cells have disappeared, prey cells are present in abundance. --> not true, mCHERRY was not completely expressed!! - measurement on saturday night, sunday, and monday of cultures in TB with/without agar (1x, 5x, and 50x) files saved with following pattern: without agar: 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. with agar: 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.
