Bjorn Millard: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
Line 3: Line 3:
Sorry about everyone who tried to go to my webpage and it was all pixelated and stuff... im new to web stuff so i'll work on it and get back to you...
Sorry about everyone who tried to go to my webpage and it was all pixelated and stuff... im new to web stuff so i'll work on it and get back to you...


==T7.1 evolutionary relaxation==
*'''Adaption protocol:'''


Basic Idea:
(1) 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


(1) Measure initial fitness of T7+
(2) 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.


(2) Measure initial fitness of T7.1
(3) Add (amount?) phage to flask and grow for 20-60 min until density of free phage is about equal to cell density


(3) Evolve T7.1 many generations
(4) A portion of the infected culture is quickly transfered to the next flask containing fresh cells


(4) Intermittently measure fitness of T7.1e's (hope to see fitness increase)
NOTE: Cultures of the continuous transfer protocol were occasionally allowed to lyse before transfer to ensure high levels of co-infection which promotes recombination


(5) Sequence T7.1e when finished with evolution and try to determine what changes in the genome increased fitness
(5) Lysates from each passage were stored




*'''Duration of adaptation:'''


'''Methods used in various evolution experiments:'''
Continue serial transfers long enough to ensure no major fitness changes occur. 


*NOTE: These methods were taken directly or summarized from the italicized paper titles below
''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.




''Gene order constrains adaptation in bacteriophage T7 - Springman et al''
*'''Media'''


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


'''The evolution:'''


*Thawed aliquots of cells were added to 125 ml flasks containing 10 ml of LB (orbital water bath at 37 -C, 200 rpm), such that the density of cells after 1 h of incubation was between 5e7 and 2e8/mL
*'''Bacterial Host:'''


*Phage were added and grown for 20 – 60 min before transfer to the next culture.
All host bacteria were BL-21 (Why not IJ1133 or BR-2???)


*The idea was to allow growth until the density of free phage reached approximately the same density as cells.


*Some strains they used chloroform treatment between each transfer, but if you transfer the phage quickly to the next flask containing fresh cells then it is not needed.
*'''Phage Strains:'''


*In addition, cultures of the continuous transfer protocol were occasionally allowed to lyse before transfer, ensuring high levels of coinfection and therefore promoting recombination.
2 strains of phage were used in these experiments:


*Lysates from each passage were stored and in some cases were used in determining when specific mutations dominated the population of phages.
(1) T7+ (wild-type)


*Each line was propagated long enough to ensure that major fitness changes were no longer occurring
(2) T7.1 alpha-beta


*Generation times generally shorten during adaptation as fitness improves, and it is consequently difficult to determine the exact number of generations during a long propagation.


*However, all lines were grown in excess of one hundred phage generations. More significantly, the impact of passage duration on evolution can be determined from the number of hours of propagation, independently of generation time (Bull et al., 2004). For example, starting at frequency of 106, a mutation that increases fitness by 1 doubling/h requires 20 h of passage to reach a frequency of 0.5 in the population, regardless of baseline fitness.
*'''Fitness Test:'''


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


*All evolved genomes were fully sequenced
'''Fitness = [log2(Nt/N0)]t'''


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


*Fitness is measured as the rate of population growth of a phage sample, represented as the number of doublings per hour (calculated as [log2(Nt / N0)]t , where Ni is the number of phage at time i , corrected for dilutions over multiple transfers, and t is measured in hours)
Nt is the number of phage at time i (i>1 hr), corrected for dilutions over multiple transfers,


*Fitness was assayed in the same conditions used for adaptation, except that low phage densities (and thus low multiplicities) were maintained during the assay. Assays lasted a minimum for 2 h (serial transfers across at least 3 flasks) and used the titer of phage at the end of the flask at 1 h as N0. In this way, new infections are largely asynchronous (and thus the phage is in exponential growth) before the first time point is taken.
t is time measured in hours.  




*'''Determination of number of phage:'''


''Experimental Evolution Yields hundreds of mutations in a functional viral genome - Bull et al''
Standard phage titering is used to determine the approximate number of phage in a solution




'''1st phase: Mutagens'''
*'''Lysis curves'''


*Cultures contained the mutagen nitrosoguanidine at 42 degrees C
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.
 
*Liquid culture was allowed to proceed to lysis, whereupon 2 microL of the lysate were transferred to another 2 mL culture of E. coli plus mutagen. 
 
*After 5 serial transfers, the phage population was plated to recover a single plaque, and a suspension of that plaque was used to initiate the next set of five liquid cultures.
 
*The plaque selection represents evolutionary bottleneck (present study used 120 liquid cultures total for each strain)
 
 
'''2nd phase: Recovery Phase'''
 
*Use E coli at 37 degrees w/o mutagens
 
*Series of passages in which a suspension of phage was added to 10 ml LB culture of cells in 125 ml flasks and grown with aeration. 
 
*Culture was then treated with chloroform and an aliquot containing at least 10e5 phage was added to the next culture
 
*Series continued for 82-115 cycles
 
*Goal was to transfer sufficient phage to maintain a moderately large minimum population size (10e5) yet transfer before high levels of multiple infection were achieved (culture was not allowed to lyse)
 
*Initial passages were conducted for 60 min before transfer, but as fitness improved it became necessary to reduce duration of each passage to 40 min because phage fitness was high enough that inoculum of 10e5 phage would overwhelm the cells within 6 min

Revision as of 16:38, 7 December 2005

I am rotating in the Endy Lab

Sorry about everyone who tried to go to my webpage and it was all pixelated and stuff... im new to web stuff so i'll work on it and get back to you...

  • Adaption protocol:

(1) 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

(2) 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.

(3) Add (amount?) phage to flask and grow for 20-60 min until density of free phage is about equal to cell density

(4) 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

(5) 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 (Why not IJ1133 or BR-2???)


  • 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.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Bjorn_Millard&oldid=10372"