T7.1/Evolution

T7.1 Evolution Protocol

NOTE: The below protocol has been adapted from various papers from the Bull and Molineux labs out of UT Austin

Adaption protocol:

• Take 0.5 mL of Klett 80 (~7e8 cells/mL) E. coli frozen aliquot (-80C freezer), thaw, and put into 10 mL LB solution in a 125 mL flask

• Let grow in orbital water bath at 37C, 200 rpm, hoping that after 60 minutes the density of cells is between 5e7-2e8 cells/mL.

• Add 1e4 - 1e7 phage to flask and grow for 20-60 min until density of free phage is about equal to cell density

• A portion of the infected culture is quickly transfered to the next flask containing fresh cells

NOTE: Cultures of the continuous transfer protocol were occasionally allowed to lyse before transfer to ensure high levels of co-infection which promotes recombination

• Lysates from each passage were stored

Duration of adaptation:

Continue serial transfers long enough to ensure no major fitness changes occur.

Springman et al. propagated 57 h, 65 h, 50.5 h (3 different strains of phage); Since generation time shortens as the phage gains fitness it is difficult to determine exact number of generations, however they state all phage were grown in excess of 100 phage generations.

Media

All media used was LB broth (10 g NaCl, 10 g Bactotryptone, 5 g Bacto yeast extract per liter)

Bacterial Host:

All host bacteria were BL-21

How to make a small bacterial overnight culture

How to make a large bacterial liquid culture

Phage Strains:

2 strains of phage were used in these experiments:

(1) T7+ (wild-type)

(2) T7.1 alpha-beta

Fitness Test:

Fitness will be measured using a doublings/hour metric as calculated:

Fitness = [log2(Nt/N0)]t

N0 is the number of phage after 1 hour, to ensure asynchronous infection and exponential growth phase;

Nt is the number of phage at time i (i>1 hr), corrected for dilutions over multiple transfers,

t is time measured in hours.

Determination of number of phage:

Standard phage titering is used to determine the approximate number of phage in a solution

Lysis curves

Lysis curves were measured by optical density of cell culture with a Klett-Summerson colorimeter. Cells were grown in a 125 mL sidearm flask to a density of 1e8 cells/mL prior to infection, at which point phage was added at a multiplicity (MOI) of ~5 and time sequential optical density readings were taken.