Lauren Honan

BIOL 550L Section 003

Experimental Design Essay #1

1. Question and Hypothesis

Question: Do various types of stressors causing cell wall damage (i.e. physical
stress, chemical stress, and environmental stress) on the gram-negative bacteria
Escherichia coli (E. coli) elicit different response mechanisms unique to the stress
type?

Hypothesis: Previous reports showed that defects in the peptide and/or glycan
linkages of the E. coli cell wall result in a cracking response such that the cell shape
can be maintained and response to antibiotic treatment results in a local bulging and
lysis response also maintaining the cell shape. Thus, it is hypothesized that (1) in
response to physical stressors such as the lysis of peptidoglycan linkages by
endolysins, the cell will respond with a cracking response, (2) in response to
chemical stress such as antibiotic treatment with penicillin, the cell will respond with
a local bulge and lysis response, and (3) in response to environmental stress such
as heat-shock, the cell will respond with increased porosity due to partial
denaturation of the peptidoglycan linkages. I predict that these three responses are
unique to the type of stressor, and all three will result in maintenance of the cell
shape.

2. Experimental Plan

To test the cell response to physical, chemical, and environmental stressors, I will
perform three separate experiments using E. coli cells. For each experiment, I will
use the imp4213 strain of E. coli noted in the literature grown to log phase in
lysogeny broth (LB) medium such that I can compare the results of my
experimentation to the results found by Huang et al. by replicating similar
experiments and altering only the type of stressor introduced to the cell. Similarly, I
will replicate their time-lapse experimental methodology to tease out the stressor-
specific differences of cell response mechanisms. Thus, for each experiment
mentioned below, I will pipette the E. coli cells onto a 1% agarose pad made with
LB. Then, the stressor will be introduced, and I will commence time-lapse
microscopy with imaging every 2 minutes. This will ensure that the cell responses
they saw and the cell responses observed in my experiments do not differ between
strains of E. coli or methodology and are unique, instead, to the type of stressor.

Physical Stressor Test: I will introduce a hydrophobic amino acid modified version
of the E. coli phage endolysin Lysep3, an endolysin previously noted in the literature
to be able to lyse the peptidoglycan linkages of the bacterial cell wall, into E. coli
cells prepared as described above to a final concentration of 1 g/mL. I will then
monitor changes to the cell wall structure over time using time-lapse microscopy and
imaging every 2 minutes.
Chemical Stressor Test: I will treat E. coli cells prepared as described above with
penicillin to a final concentration of 1 g/mL. Penicillin’s known mechanism of action
is to inhibit the cross-linkage of peptides on the cell wall by binding to
transpeptidases, therefore limiting cell wall synthesis. I will then observe the
changes in the cell shape and cell wall structure over time using time-lapse
microscopy with images taken 2 minutes apart.

Environmental Stressor Test: I will slowly expose E. coli cells prepared as

described above to increasing temperatures from room temperature (25C) leading
up to 110C, the temperature noted in literature at which E. coli cells are completely
denatured. The cells will be heated in a hot water bath slowly increased from 25C to
110C to monitor the temperature over time. Throughout this increase in
temperature, I will monitor changes to the cell wall structure using time-lapse
microscopy taking images every 2 minutes, specifically paying attention to the time
period of partial denaturation before reaching 110C.

3. Anticipated Results and Interpretations

I anticipate that the physical stressor test will result in time-lapse microscopy
displaying the cracking cell response. This is because the bacteriophage will
produce localized breakages in the peptidoglycan linkages of the cell wall, and the
cell will crack around the center of this localized damage. This cracking response
allows the rest of the cell to maintain its cell shape, critical to cellular function
regulation. I anticipate that the chemical stressor test will result in time-lapse
microscopy showing the formation and lysis of a bulge. This bulge is a localized
response to accumulation of cell damage over time from the passage of penicillin
into the cell membrane. Over time, the cell will crack around the bulge and
eventually lyse it, returning the cell to its desired cell shape. Finally, I anticipate that
the environmental stressor test will result in time-lapse microscopy showing a
uniform distribution of peptidoglycan defects on the cell wall because the increase in
temperature will lead to partial denaturation of the peptide linkages. This partial
denaturation will lead to increased pore size of the membrane in the areas in which
the damage occurred, and shrinkage of the pore size of unaffected areas. This
allows the cell to maintain its shape in response to the damage. Ultimately, I
anticipate the three types of stressors being investigated to produce unique damage
control responses in order to maintain cell shape and structure as best as possible.
Physical stressors will result in cell wall cracking response, chemical stressors will
result in bulge and lysis response, and environmental stressors will result in
increased porosity of the cell wall.