T7.1/Evolution

Why evolve T7.1?

The refactoring process of the T7 phage to produce the T7.1 phage was a success in the fact that the un-overlapping of the alpha and beta sections of the T7 genome still produced a viable phage. Though it is viable, it is less fit than the wild-type T7 phage with respect to growth rate. This observation gives us an opportunity to use short term evolution as a debugging tool to let nature help us discover what was disrupted during the refactoring process that causes reduced fitness.

T7.1 Evolution Protocol

NOTE: The below protocol has been adapted from various papers from the references listed below

Adaption protocol:

Taken from Springman 2005:

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

• The culture was placed in orbital water bath at 37C, 200 rpm for 60 min where cells were grown to a density between 5e7-2e8 cells/mL.

• 1e4 - 1e7 phage was added to the flask and grown for 20-60 min until density of free phage was approx. equal to cell density

• A portion of the infected culture was 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

• The above steps were repeated ~4-5 times a day

• At the start of each day, the stored phage stock from last previous day's passage was used as starting phage for current day's passages

Duration of adaptation:

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

Springman 2005 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

LB broth (10 g NaCl, 10 g Bactotryptone, 5 g Bacto yeast extract per liter)

Bacterial Host:

Ideally we wanted to find a bacterial host in which the T7+ (WT) had minimal nucleotide evolution, yet still provided selection pressures for the T7.1.

Springman 2005 notes:

"Wild-type T7 has not been extensively grown in the laboratory since its isolation in 1945 (Studier, 1969, I. J. M., unpublished). Its usual hosts are E. coli B derivatives, rather than the K-12 strain used here, and the usual temperature of growth is 30 -C, rather than 37 -C. It was therefore expected that the T7+ was not well adapted to our culture conditions. " <-- They used IJ1133

Heineman 2005 notes : "The T7+ had already been passaged in this lab condition on IJ1133 for some time and was passaged for 20.5 more hours to create a control for the expected fitness increase in the abscence of any genomic defect"

Host chosen:

BL-21

Reason: It is supposedly the usual host but we still performed adaptation procedures for T7+, as in Heineman 2005, to confirm it has reached its final steady state of adaptation

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

Determination of number of phage:

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

Measuring Phage Lysis Curves

Taken from Chan 2005 - Supp. Materials:

1mL containing 2E8 cells of BL21 was infected at a MOI of 5 and 200uL of the resulting mixture was loaded per well into a 96 well ViewPlate (Packard) at 30°C. Mineral oil was layered into each well and the OD was monitored at 30°C with agitation by a Wallac Victor2 plate reader (Perkin-Elmer). The half-lysis time was taken at the time when the absorbance of a culture equals the average of its absorbance at (i) the time of infection and (ii) the end of lysis.

Fitness Test:

Fitness was 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.

References

• Bull JJ, Badgett MR, Rokyta D, Molineux IJ, (2003) Experimental evolution yields hundreds of mutations in a functional viral genome. J Mol Evol 57:241-248
• Bull JJ, Badget MR, Wichman HA, Huelsenbeck JP, Hillis DM, Gulati A, Ho C, Molineux IJ, (1997) Exceptional convergent evolution in a virus. Genetics 147: 1497-1507
• Chan L, Kosuri S, Endy D, (2005) Refactoring bacteriophage T7. Molecular Systems Biology 10.1038/msb4100025
• Cunningham CW, Jeng K, Husti J, Badgett M, Molineux IJ, Hillis DM, Bull JJ, (1997) Parallel molecular evolution of deletions and nonsense mutations in bacteriophage T7. Mol. Biol. Evol. 14(1): 113-116
• Garcia LR, Molineux IJ, (1996) Transcription-Independent DNA translocation of bacteriophage T7 DNA into E. coli. J of Bacteriology p. 6921-6929
• Heineman R, Molineux IJ, Bull JJ, (2005) Evolutionary robustness of an optimal phenotype: Re-evolution of lysis in a bacteriophage deleted for its lysin gene. J Mol Evol 61:181-191
• Springman R, Badgett MR, Molineux IJ, Bull JJ, (2005) Gene order constrains adaptation in bacteriophage T7. Virology 341 141-152
• Rokyta D, Badgett MR, Molineux IJ, Bull JJ, (2002) Experimental genomic evolution: extensive compensation for loss of DNA ligase activity in a virus. Mol. Biol. Evol. 19(3): 230-238