---+ Measuring Lysis Timing of T7 Phage ---++ Reagents / Materials: * Phage lysate * see [[ProtocolPhageLysate][Preparing Phage Lysates]] * Overnight stock of permissive bacterial host * Make sure to grow in presence of 250uM nsAA if using nsAA-RS strains * [[stub][LB Media w/ appropriate supplements and antibiotics]] * 250mL flasks * *For plating phages:* * LB Plates with w/ appropriate supplements and antibiotics * LB Top Agar (LB + 0.8% Agar) * Test tubes * 55C Water bath or heat block * 37C Incubator ---++ Procedure: 1 Add 500uL of an overnight culture of the E. coli host to 9.5mL of LB in a 250mL flask 1 Allow to grow for 1h @ 37C, 200RPM until culture density is 1-2e8 cells / mL (OD ~ 0.5 ?) 1 Add 1e6 phages and incubate 5min without shaking at 37C (may need to be shorter for some strains?) * Start a timer counting up here, will need to take further timepoints fairly rapidly 1 Transfer 100uL of culture to fresh 10mL (1000x dilution) of LB pre-warmed to 37C (may need to adjust volumes?) 1 At 5.5min from start of infection, take a 1mL sample, quickly take 10uL for a titer, then add 50uL chloroform to the sample, vortex, then take a second 10uL titer 1 At 6.5min from start of infection, take another 1mL sample, quickly take 10uL for a titer, add 50uL chloroform to the sample, vortex, then take a second 10uL titer * These happen pretty quickly, make sure tubes &c for titers are set up beforehand 1 Take titers at 15.5, 16.5, and 17.5 min after chloroform treatment * Only concerned w/ free phage here, so no need to titer before chloroform treatment for these. Have tubes w/ chloroform ready before samples are collected. 1 To calculate burst size: * First calculate the number of initially infected cells Ni = (early timepoint titer *BEFORE* chloroform treatment) - (early timepoint titer *AFTER* chloroform treatment). Chloroform treatment before end of the eclipse period results in non-viable infections, so only free phage are counted. Because phages are limited to single infections (by large excess of host cells), number of infected cells is just the total infective centers minus free phages. * Burst size = (average titer of *free* phages at late timepoints) / Ni ---++ References: [1] Heineman, Richard H, and James J Bull. "Testing Optimality with Experimental Evolution: Lysis Time in a Bacteriophage." _Evolution_ 61, no. 7 (2007): doi:10.1111/j.1558-5646.2007.00132.x.
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Topic revision: r1 - 2017-06-15 - ColinBrown