This document summarizes research into using Colicin-E7 to prevent urinary tract infections (UTIs). Colicin-E7 is a bacteriocin produced by some E. coli strains that kills other related strains. The goal is to stabilize purified Colicin-E7 in solution so it can be used to kill E. coli bacteria and prevent UTIs in women in the Army. Researchers incubated E. coli cultures to produce Colicin-E7, then purified and tested it in solutions with and without various components like ethanol and Tween 20. Drop tests showed the supernatant containing Colicin-E7 was most effective at killing E. coli, while activity of the retentate decreased

1. UNCLASSIFIEDUNCLASSIFIED
Using Colicin-E7 to Prevent Urinary Tract Infection
Meghan Halpin; Mentors Robert Stote and Caitlin Barrows
Natick Soldier Research, Development & Engineering Center; Warfighter Directorate
Introduction
Urinary Tract Infection (UTI) is a common problem for
many women during their lifetimes. In fact, about one in
five women are expected to experience UTI during
adulthood. The infection is caused most commonly by E.
coli, which is responsible for 70-95% of all UTIs. Several
factors can influence UTI development, including genetic
predisposition, lifestyle and behavior, and pregnancy.
There are many strains of E. coli, several of which
produce bacteriocins. Colicins are one type of
bacteriocin, and are long proteins produced by some
strains of E. coli to kill related strains in competition. We
are focused on colicin-E7. While colicins E1 through E9
exist, E7 has proven to be the most effective against UTI
causing bacteria and displayed the least amount of
antibiotic resistance. The goal of this project is to
stabilize colicin-E7 in a purified form when it is in a
solution. The colicin can be used to kill off bacteria that
cause UTI for women serving in the United States Army.
Approach
 Two 100 mL cultures were inoculated with E7 and then
incubated overnight with agitation. They were used the
next day to inoculate two 900 mL flasks of Luria Broth.
These flasks were then incubated for one hour and the
optical density was recorded (about 0.300 abs).
 After one hour, the two flasks were then induced with
mitomycin C and were incubated for an additional
three hours.
 The cells were removed by centrifugation. The product
is called supernantant, and was filtered using
tangential flow. The result is called the retentate, which
was also spun down by centrifugation.
 The FPLC gathered fractions. Omissions tests were
then prepared using ethanol, Tween 20, glycerin,
catalase (from the glycerin), EDTA, and sodium citrate.
The omissions are based off of a wipe that prevents
infections in cows. We prepared a control and 6
additional tests, each missing one of the components.
 The pooled fractions from the HPLC were then
distributed among the omissions and drop tested.
Acknowledgements
 UMass Lowell Co-Op Scholars
Program
 Adrianna Morris
 Natick Soldier Research,
Development & Engineering
Center
 John Player, Robert Stote &
Caitlin Barrows
Results
Every week, we performed multiple drop tests with
supernatant, retentate, and omissions tests. We made
overlays for the plates with Luria Broth soft agar and
either 54398 or DH5α, which are two strains of E.coli.
When deciding what to drop, we included the current
week’s supernatant, retentate, and omissions tests, but
we also looked at pictures of plates from previous weeks
and included what was still showing activity. For controls,
we also dropped Luria Broth and bleach in the bottom
right of each plate. Luria broth does not kill anything,
while bleach will kill any bacteria present.
Figure 1. Active fractions in stabilizing solution dropped
on Day 8 onto a soft agar overlay of DH5α.
Figure 2. Supernatant dropped on Day 8 on soft agar
overlay of DH5α
Discussion & Conclusions
 For the omissions, we have gathered that as purity
increases, stability decreases. In order for the
solution to be put into a wipe, the solutions needs to
be stable. The omissions tests mimic the
components of the wipes used for animals, and are
important for distinguishing what components are
crucial, which appear to be ethanol and Tween 20.
When the solution is dropped onto an overlay, lack of
activity shows that the missing component is crucial
to the solution in its entirety.
 We also have found that the supernatant seems to
be more effective than the retentate. Usually after
about one week, the retentate looses activity. It is
suspected that this could be due to either
degradation or aggregation of the protein.
References
Carr, J. H. (2016). E. coli bacteria. Retrieved from
http://phenomena.nationalgeographic.com/2016/06/01/its-crucial-the-new-superbug-
was-in-a-urinary-tract-infection/
Cascales,E. (2007). Colicin Biology. Microbiology and Molecular Biology Reviews,
71(1), 158-161.
Foxman, B. (2000). Urinary Tract Infection: Self-Reported Incidence and Associated
Costs, AEP, 10(8), 509-515.
Figure 3. Tangential flow retentate dropped on Day 8 on
soft agar overlay of DH5α.
Figure 4. This plate is a drop test from our omissions
samples. On the left is omissions test 1. The solution
was dropped out to 6 dilutions. The missing component
of this omissions test is ethanol. On the right is
omissions test 2, which is missing Tween 20 from the
solution. This was also dropped out to 6 dilutions.
Since we remove one component from each of the six
omissions tests, we are able to tell which components are
crucial, and which are not. Using the plate above as an
example, we are able to predict that ethanol and Tween
20 are vital components to the solution.