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John Goddard – Research Summary 2012-2013

Section 1: Introduction

In the context of eventually characterizing the effect lysis time variance has on mutation fixation

in phage, my project aimed to determine how various point mutations affect lysis time and burst

size in phi X 174. Due to its small genome, short generation time and the fact that its natural host

is the extremely well understood Escheria coli, phi X 174 is an ideal phage for studying the

effects of point mutations. Phi x 174 lyses host cells with the product of a single gene, the E

gene. Research was conducted using two sets of mutant phage that had been assembled for

previous work in the lab. The first is composed of mutants that contain mutations in the promoter

region for the E gene and the second is a set of mutants that have mutations directly in the E

gene, and the mutants from both sets have been shown to affect lysis parameters.

Two primary experiments were conducted to determine lysis characteristics in the phage.

Over the summer, one-step growth experiments (so called the “Cranky Dragon” experiments)

were conducted with cultures of E. coli infected with phi X 174. Throughout the academic year,

the primary experiment focused on directly measuring burst size and lysis time by putting an

infected cell on a 22 micron filter and repeatedly washing the filter with LB at set time intervals.

This document will focus primarily the latter of these two experiments (and the difficulties

associated with conducting it at room temperature), but more information on the one-step growth

experiments can be found in Appendix A.

The Phage: The mutants in the first set (pos4B, pos5 and pos6) have mutations directly in the E

gene and were first characterized by Bernhardt et al. in 2002. The second set of mutations

(mut319, mut321, mut323 and mut324) are point mutation in the promoter region for the D gene

(the E gene is embedded in the D gene), which is located in the last few nucleotides of the C

gene. They were first discovered by Brown et al. in 2010. The mutations and the labels used for

them in the lab are shown in table 1 below.

Genotype Label in the

Lab

Nucleotide

Changes

Amino Acid

Changes

Known effects

on lysis in E.

coli

pos4B pos4B several R3

H and

L19

F Same as wt

pos5 pos5 several L19

F +10 min

pos6 pos6 several R3

H Same as wt

mut319 D G319

T V63

F(C protein) +2 min

mut321 C T321

C none +2 min

mut323 A A323

G N64

G(C protein)

+2 min

mut324 B C324

T none +2 min

Table 1. Information on the phi X 174 mutants. The “label in the lab” section corresponds to the ways the freezer stocks are labeled. The “known effects on lysis” were taken from the studies that identified the mutants. This information was adapted from Chris Baker 12’s thesis.

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Section 2: Experiments and Results

Direct analysis of phage lysis time and burst size: The primary experiment that was conducted

(with varying degrees of success) this year was one adapted from a 1963 California Institute of

Technology study that aimed to determine whether phage lysis was a discrete or continuous

process (Hutchinson & Sinsheimer, 1963). Using a system of filtration and washing, the protocol

has the power to determine the lysis time and burst size of single lysis events with little to no

background noise. Mutant C and the pos6 mutant were focused on most heavily as those are the

two mutants that showed a significant difference in lysis parameters in Chris Baker’s analysis.

Methods: First, the day before the experiment is to be conducted, an overnight of E. coli is

created by adding one colony of stock E. coli C to 10 mL of lennox broth (LB) media with a

CaCl2 concentration of 2 millimoles per liter. The overnight is grown for 16-24 hours in a 37˚C

incubator. To initiate the experiment 10^6 phage were added 10^8 E. coli cells in 1 mL of liquid

LB media in a test tube. The test tube was placed in a shaker in the 37˚C for 8 minutes. After this

point, serial dilutions were performed, and a volume appropriate for the number of desired

infected cells (generally between 1 and 20 infected cells) was applied to the 22 micron filter in a

filter eppendorf tube. These tubes were then centrifuged immediately (exactly 10 minutes after

the phage and cells were added to the LB). The filters were then washed with 500 μL of LB and

centrifuged every 3 minutes for an hour. Washes were placed on ice and then plated at the

conclusion of the experiment. 4-8 replicates were used for each experiment. LB was kept at

37˚C. Different methods were used to try and keep the infected cells at 37˚C. The primary ways

of doing this were placing the filters in a heat block in between washes and keeping them

spinning in the centrifuge (set at 37˚C) in between washes. Plating was done by adding the 500

μL sample and 200 μL of bacteria to 3 mL of 42˚C top agar (also with a CaCl2 concentration of 2

millimoles per liter). Plates were incubated for 4-6 hours at 37˚C and then scored for plaque

forming units (pfus). During some points of the year stationary phase E. coli (concentration ~109

cells/mL) were used and at others stationary phase bacteria that had been diluted 30x and

regrown to a concentration of 600 OD (mid exponential phase; concentration ~108 cells/mL)

were used. There were no discernible patterns of differences that arose from using exponential

phase cells instead of stationary phase cells.

The use of synchronized E. coli cells: Initially, E. coli cells were synchronized using the HFB-

1/HFB-2 method used for Cranky Dragon (see Appendix A) in order to ensure that they were at

the same point of the cell cycle. This step was eventually deemed unnecessary for preliminary

runs of the experiment, but it could be worth further exploring if the experiment is ever being run

with consistent success, as it would eliminate effects that cell cycle variability could have on

lysis time and burst size.

A note on temperature: Ideally, this experiment would be performed in a room where a

temperature of 37˚C was maintained. Instead, the experiment was run trying to keep the

temperature as close to 37˚C as possible as described above, as well as at room temperature. A

more detailed explanation of the effects of these different conditions is given later on.

Representative Results: The expectation for this experiment was that burst events would be seen

at certain time points (where plates would have upwards of 100 pfus) and that most other plates

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would show no phage (other than maybe a few residual phage after a lysis event that took

multiple washes to remove from the filter). Typical results, however, broke down into three

major categories. In the first, results met these expectations (a single lysis event with a high pfu

count; see figure 1). In the second type of results, what appeared to be a lysis event (a non-zero

Figure 1. A run of the experiment that produced "ideal results". This particular run was conducted by putting approximately 5 exponentially growing cells infected with wild type phi X 174 phage on the filter. There was an attempt to maintain temperature at 37˚C throughout this run. See composite results spreadsheet 11-1 for this data.

pfu plate from a time point far enough from the starting time to feasibly be caused by a cell

lysing and releasing phage) occurs but the pfu count is low (

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whether or not there was a problem with adsorption and whether temperature had a significant

effect on experimental results.

The sticky-filter hypothesis: It was proposed that there was a possibility burst-numbers were low

because a certain proportion of phage were sticking to the 22 micron filters. In order to test this,

two equal volumes of diluted phage stocks were taken. One of the samples was plated directly,

and the other was washed through a filter and then plated. Results showed no significant

differences in terms of pfus (spreadsheet 2-7). In order to further ensure that the filters behaved

as expected, a sample of LB containing a known amount of bacteria was washed through one of

the 22 micron filters. The wash was then plated to look for bacterial growth, and there was none.

These results indicate that the filters behave as expected, allowing phage but not cells to pass.

Second generation phage adsorption: Another hypothesis as to why burst counts were lower than

expected was that second generation phage (those released after a burst event) would attach to or

infect other cells on the filter before being washed through. This was considered a viable

possibility, because with MOI values ranging from .1 to .001, there were 10 to 1000 times as

many cells as phage on the filters. However, rough area-based calculations showed that these

cells would occupy no more than 1% of the surface area of the filter. To verify that any such

effects would be small experimentally, simulated bursts were conducted whereby standardized

volumes of diluted phage were placed on filters with known numbers of bacteria and washed at

either 1,2,3,4 or 5 minutes (to simulate the amount of time that might pass between a burst and

wash). The wash was then plated with the standard E.coli and top agar procedure. Neither time

nor number of cells had a significant effect on the number of phage that passed through the filter

(at least not one that could explain cutting the number of phage that pass through the filter by

half or more; see table 2, below).

Time/Cells 0 cells 10^3 cells 10^6 cells

1 82 77 85

2 85 80 72

3 79 75 79

4 80 70 77

5 79 83 71

Mean: 81.5 75.5 78.25

Table 2. Results from a simulated burst experiment where a particular volume of diluted phage was washed through a filter with a known number of cells on it. The results revealed that a scenario whereby 50% + of the second generation phage were being adsorbed by cells on the filter is highly unlikely.

Phage adsorption analysis: After trying a few runs of the main experiment at room temperature

(with the idea that doing it at a consistent temperature was better than having multiple

temperatures between the heat block, centrifuge and room, and that slowing things down at room

temperature would amplify differences between mutants), and seeing an increase in the number

of runs that looked like cases of low adsorption (a high number of pfus on the first wash and very

few thereafter), a new experiment was conducted to determine the number of phage that were

being adsorbed during the initial infection period. Phage and bacterial cells were added to 5 mL

of LB (at various MOIs), and they were mixed (with multiple replicates at both 37 degrees and

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room temperature). Samples were taken every three minutes and then centrifuged through a 22

micron filter to isolate any unadsorbed phage. These samples were then plated and the plates

were incubated and scored for pfus in order to determine the number of unadsorbed phage.

Typical results are shown in figure 3, below:

Figure 3. A plot of phage adsorption versus time at MOIs of .1 (blue) and .001 (red) and a temperature of 37˚C. Each plot is a composite of 5 replicates. The replicates were normalized by dividing the count at a given time by the count at time 0. These values were averaged across the replicates. The y axis was then log transformed. See composite results spreadsheet 2-25 for this data.

Regardless of temperature or MOI, max adsorption was about 50% of phage. While this is

consistent with the results that had been seen with the primary experiment (where ~50% of the

total phage often washed through at the first time point), it is an order of magnitude plus away

from work being done in the lab by Mike Bieszard where phage adsorption was often 90% or

higher. These results were consistent across the pos6 mutant, the C mutant and the wild type

strain. In response to these results, a run of the original experiment was conducted where

concentration of phage and cells were maximized during the infection process (106 phage were

added to 1mL of stationary phase E. coli. This had little effect. Again, nearly one half of the

phage put on the filter washed through on the first wash.

As noted earlier, this adsorption analysis was performed both at 37˚C and room

temperature (21-22˚C). In later trials, plates were incubated at room temperature in order to

maintain a consistent temperature throughout the experiment. When this was done, no plaques

formed. This was consistent across the wild type phage and both mutants (pos6 and C).

Exploring the effects of incubation temperature on plaque count: To determine if these plates that

were showing no visible plaques where actually forming plaques that couldn’t be seen, set

volumes of phage of all three target strains were plated in top agar and incubated at room

0.1

1

10

0 5 10 15 20 25 30 35

No

rmal

ize

d C

ou

nt

(lo

g sc

ale

)

Time (minutes)

Normalized phage count vs time

Low MOI

High MOI

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temperature. When no visible plaques formed, the layer of top agar (and the lawn of bacteria) of

each of plates was scraped off, put in LB solution, vortexed and centrifuged. The supernatant

was then plated in top agar, and these plates were incubated at 37˚C. If non-visible plaques were

forming, the expectation is that a high number of pfus would be seen in this round of plating.

Instead, phage counts were observed that were consistent with the number of phage that would

be expected to be in the volume put on to the original round of plates. This supports the

hypothesis that (at least one step of) the phage-bacteria interaction was stopped at room

temperature.

It is also worth noting that some plates were left out at room temperature and then put in

the 37˚C incubator. If the plates were put in the incubator before there was a visible lawn of

bacteria, plaques would form. If there was already a lawn, they would not.

To find out where the “threshold temperature” for plaque formation was, and to

determine whether the number of plaques that forms is continuously temperature dependent (as

opposed to a more discrete phenomenon where the same number of plaques forms regardless of

temperature as long as the temperature is above the plaque forming threshold), standardized

volumes of the 3 phage types of were added to top agar and plated. These plates were incubated

at a variety of temperatures (22˚C, 26˚C, 28˚C, 31˚C and 37˚C; see figure 4).

Figure 4. Plaques vs. temperature for a standard volume of wild type and pos6 phage. The analysis was also conducted with the C mutant, but counts were so low that they were discarded. See composite results spreadsheet 4-15.

This analysis revealed a relationship between incubation temperature and the number of plaques

that formed (R2

values of .7976 and .7638 for the wild type and pos6 strains, respectively). A

repeat of the experiment showed similar results, but, ideally, the experiment could be repeated

multiple times to confirm this phenomenon.

y = 1.6144x - 7.8801 R² = 0.7976

y = 1.4017x - 0.1426 R² = 0.7638

0

10

20

30

40

50

60

25 27 29 31 33 35 37 39 41

pfu vs. Temp

WT

Pos6

Linear (WT)

Linear (Pos6)

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Discovering a mutation that allows for plaque formation at room temperature: An effort was

made to create a strain of phage with a mutation that allows for plaque formation at room

temperature. In order to do this, 100mL aliquots of highly concentrated phage stock were plated

in top agar and the plates were incubated overnight at room temperature. Had a plaque formed, a

stock would have been created from it, but no such plaque did form. This stock could have been

sequenced to determine the mutation that allows for plaque formation at room temperature. To

maximize the possibility of such a mutation, high titer stocks of phage were created by

harvesting lacy lawns, filtering and adding glycerol. These stocks generally had a maximum

concentration between 108-10

9 phage/mL. Finding a way of increasing this concentration could

increase the likelihood of finding a mutation that allows for room temperature plaques.

Incidentally, while conducting this search for a novel mutant, it was discovered that the

pos5 mutant can form plaques at room temperature. This implies that mutations to the E gene

(like the L19

F mutation in pos5) can have an influence on the temperature dependence of

plaquing in phi X 174. To further investigate pos5, the original experiment was conducted with it

at room temperature. The last time point looked like a potential lysis event (still a low burst size

of 51, figure 5). Repeat experiments would need to be performed for longer periods however, to

provide conclusive evidence that the experiment works successfully with pos5 at room

temperature.

Figure 5. Original experiment run at room temperature with pos5 (one replicate with ~1 infected cell on the filter and the other with ~10). The last time point shows what looks like a possible lysis event in the 10 phage replicate. See composite results spreadsheet 5-5.

Conclusions: While no additional claims can be made about the two mutants (pos6A and C) that

showed significant difference in Chris Baker’s work, the work done here implies that

temperature is an important factor in phi X 174/ E. coli infection dynamics. Any further attempts

to characterize differences between these mutants will need to be done at a temperature that

allows for the infection to cycle to occur (through use of heat blocks, heated rooms, etc…), or

they will need to use mutants like pos5 that show activity at room temperature. Next steps might

include continuing to search for novel mutant that can form plaques at room temperature and

determining where precisely the infection cycle slows down (adsorption vs. replication vs. lysis,

0

10

20

30

40

50

60

0 20 40 60 80 100 120

10 Phage

1 phage

8

etc…). Further analysis could also be performed to determine the behavior of pos5 at room

temperature.

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References

Baker, C. 2012. Estimating the Influence of Mutations on Phage Life Histories Using a Single-

Phage Assay. Brown University Department of Ecology and Evolutionary Biology

Honors Thesis.

Bernhardt, T. G., Roof, W. D., & Young, R. 2002. The Escherichia coli FKBP-type PPIase SlyD

is required for the stabilization of the E lysis protein of bacteriophage ΦX174. Molec.

Microbiol. 45: 99-108.

Brown, C. J., Zhao, L., Evans, K. J., Ally, D., & Stancik, A. D. 2010. Positive selection at high

temperature reduces gene transcription in the bacteriophage ΦX174. BMC Evol. Biol. 10:

378.

Hutchinson III, C. A., & Sinsheimer, R.L. (1963). Bacteriophage Release by Single Cells of phi

X 174-infected E. coli. J. Mol. Biol. 7:206-208.

Hyman, P., & Abedon, S. T. (2009). Practical methods for determining phage growth

parameters. In Bacteriophages (pp. 175-202). Humana Press.

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Appendix A: Single Step Growth Assays (Cranky Dragon)

Over the summer, research focused on generating single step growth curves for the various

mutants. To do this, a certain number of phage and exponential phase E. coli cells (MOI = .01)

were added to 10 mL of LB. These cells had been synchronized through a starvation procedure

where they were washed three times HFB-1 solution and once with HFB-2. After each wash, the

cells were centrifuged, and after all 4 washes they were resuspended in LB via vortexing. Serial

dilutions were made such that there were three flasks: the original, a 10x dilution and a 100x

dilution. These flasks (containing phage and cells) were placed in the shaker in the water bath,

which was kept at 37˚C. 50 μL samples were taken from the three flasks periodically according

to a preset schedule (early samples were taken from the original flask, and later ones were taken

from the dilution flasks). These samples were put on ice (in a pcr plate on ice) and then at the

end of the experiment plated in 3mL of 42˚C top agar with 200 mL of exponential phase E. coli.

Typical results showed a low number of phage initially, a rapid growth phase during which time

lysis events were occurring, and then a plateau after this first wave of bursting occurred (figure

6).

Figure 6. Typical results from a Cranky Dragon experiment. The above analysis was done with two replicates of the D mutant (Red and Blue) and one replicate of the C mutant. The shift of the C curve to the right of the D curves indicates that on average lysis events occurred later in the C mutant.

Analysis of Cranky Dragon data: A rough sense of the mean lysis time (at 37˚C) can be gleaned

by looking at the midpoint of the time period when phage concentration is rapidly increasing.

The slope of that increase can give insight into the variance in lysis time (are most lysis events

happening over a 5 minute period or a 10 minute period?, etc…) Burst size can be inferred from

the difference between the original concentration and final concentration. However, all of these

measures have inherent noise that arises from dealing with factors like unadsorbed phage. To

90

900

9000

90000

0 10 20 30 40 50

Ph

age

co

un

t (l

og

tran

sfo

rme

d)

Time (minutes)

Adjusted phage count vs. time

D2 (1)

D2(2)

C3(1)

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more carefully estimate lysis parameters from this data, careful statistical measures would have

to be taken. For a starting point on this analysis, refer to Hyman & Abedon, 2009.

Appendix B: Where to find things

Phage Stocks:

Primary phi X 174 mutant stocks:

Location: Yellow box on the top shelf of the -20˚C freezer on the right side (marked with

a red arrow in the picture below). The label says “phi X 174 mutants”.

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In the Box:

Each stock is labeled with the name of the mutant. The letters after the names of the pos mutants

(i.e. pos5 “A”) indicate replicates, as do the numbers after promoter mutants (i.e. 37˚ C1, C2 and

C3 are replicates of each other). WT is the wild-type. Some of the stocks have an up arrow next

to a T and others have a down arrow next to a T, which indicate high titer and low titer,

respectively. A list of the most recent titers is shown below.

Stock Titer

WT A 1.5 x 108

A1 9 x 106

B1 4 x 106

C3 5.4 x 106

D2 8 x 106

pos4B B 1.6 x 108

pos5 A 7 x 107

pos6 A 1 x 108

Low-titer WT 4.3 x 103

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Replicate high titer stocks made by Mike Bieszard and John Goddard: Additional high titer

replicate stocks can be found in the green box (labeled M. Bieszard) on the second shelf of the

door of the -20˚C freezer (pictured below).

E. coli C: The stock of E. coli C can be found on the bottom shelf of the -80˚C freezer in a box

labeled “Heat Sensitive E. coli”.

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Appendix C: A guide to the data spreadsheets

Composite2012-2013.xls:

All spreadsheets contain a brief description of the experiment they are associated with

underneath the data. For spreadsheets of data from the original experiment, the top column for a

replicate is in the format “mutant-stock (expected infected cells on the filter)”. I.e. WT-A (10)

Means that it was the A replicate of the wild type and that the goal was to put 10 infected cells

on the filter. Controls for the experiments are not shown unless they were notable.

The sheets are in chronological order. Useless data was omitted.

9-27 thru 12-4: The original experiment conducted at 37˚C. 10/25 is an example of low

adsorption where there were only phage on the plates from the first time points.

2-1: Original experiment at room temperature. Titers were higher than expected, but the data

could be used to determine the expected range for lysis times.

2-7: Experiment to determine whether or not phage were sticking to the filters.

2-15 thru 2-25: Experiments to determine the percentage of phage that get adsorbed.

3-1 & 3-14: Original experiment at room temperature with an excess of cells. Possible lysis

events had low burst numbers.

4-5 & 4-15: Results from incubating plates at different temperatures.

5-5: Original experiment with pos5 at room temperature.

5-12: Simulated bursts with different concentrations of phage on the filters.

CDresults1.xls: This is the data from the Cranky Dragon experiments. Sheet1-sheet5 look at

mutant D. Sheet6 and sheet7 contain mutant C data as well. The important columns are

“adjusted” (which combine the data from the 3 different flasks) and “filtered” (which remove

unreliable low count points from the “adjusted” column. Timesheet is the most up to date

sampling schedule. A1-E4 correspond with the well of the pcr plates samples were collected in.

Times are in the column of the time flask for the sample taken at that point.