1. Sensing White Nose Syndrome in Bats
with the crea5on of an EnvZ-Tar chimera protein in Escherichia coli
Aus5n Hall, Hannah Kim, Margaret Pitzer
Research Advisor: Dr. Westenberg
Department of Biological Sciences,,, Missouri University of Science and Technology
Project Description
The fungus, Pseudogymnoascus destructans, causes White Nose
Syndrome, which is devasta5ng the North American bat
popula5on. More and more researchers are working toward
comba5ng the deadly disease and restoring the number of bats
found in the wild. The bats affected by White Nose Syndrome are
a viable asset to the United States Agricultural Industry, and so, a
remedy needs to be found. Our aPempt at an answer uses a
synthe5c biology approach. It is expected that an E. coli strain
able to move towards changes in salinity will move to the source
of the fungal infec5on, as muscle fibers and collagen of bats get
degraded via hydrolysis by the invading pathogen, P.destructans.
Consequently, the osmo5c pressure in bat’s 5ssue would increase
as water is being used to break collagen. Hence, using this trait,
along with a strain that produces a fungista5c compound, to slow
or even stop the infec5on while the bat is in hiberna5on.
Genes Involved
Tar Gene - Chemotac5c-signal transducers respond to changes in the concentra5on
of aPractants and repellents in the environment, transduce a signal from the outside
to the inside of the cell, and facilitate sensory adapta5on through the varia5on of
the level of methyla5on
Tar Gene’s func-on = Chemoreceptor
EnvZ Gene- Member of the two-component regulatory system EnvZ/OmpR involved
in the regula5on of osmoregula5on. EnvZ func5ons as a membrane-associated protein
kinase that phosphorylates OmpR in response to environmental signals.
EnvZ Gene func-on = sensing changes in osmo-c pressure
Project Goals
• Gain a deep understanding for White Nose Syndrome at
a microbiological level.
• Create a synthe5c biology design for a chemotaxis
sensi5ve bacterial cell.
• Design a DNA primer in a living bacteria (E.coli) by
analyzing gene sequence found in nature.
• U5lize known extrac5on and biological methods to
insert genes into our host E.coli backbone
Bacterial Design Procedure
Hypothesis: E.coli containing the synthe5cally designed transmembrane
chimera EnvZ-Tar protein would sense and move towards the change in salinity
(osmolarity difference P.destructans creates), indirectly sensing the fungus.
Results/ Discussion
References
1. O’Donoghue, A. J., Knudsen, G. M., Beekman, C., Perry, J. A., Johnson, A. D., Derisi, J. L., . . . Bennett, R. J. (2015). Destructin-1 is a collagen-degrading endopeptidase
secreted by Pseudogymnoascus destructans , the causative agent of white-nose syndrome.Proceedings of the National Academy of Sciences Proc Natl Acad Sci USA,
112(24), 7478-7483.
2. Lorch, J. M., Muller, L. K., Russell, R. E., O'connor, M., Lindner, D. L., & Blehert, D. S. (2012). Distribution and Environmental Persistence of the Causative Agent of White-
Nose Syndrome, Geomyces destructans, in Bat Hibernacula of the Eastern United States. Applied and Environmental Microbiology, 79(4), 1293-1301.
3. Fenton, M. B. (2012). Bats and white-nose syndrome. Proceedings of the National Academy of Sciences, 109(18), 6794-6795.
4. White Nose Syndrome of Bats Fact Sheet | Washington Department of Fish & Wildlife. (n.d.). Retrieved March 30, 2016, from http://wdfw.wa.gov/conservation/health/wns/
5. Yoshida, T., Phadtare, S., & Inouye, M. (2007). The Design and Development of Tar-EnvZ Chimeric Receptors. Methods in Enzymology Two-Component Signaling
Systems, Part B, 166-183.
Introduction/ Background
White-nose syndrome (WNS) is a disease that has a dis5nc5ve
fungal growth on the muzzle and the wings of the bat. While
hiberna5ng, the fungus breaks down the bat’s skin membranes
and depletes the fat storages needed to survive. The disease
started in the Europe and spread to the caves of North America
on the spelunking gear. In reac5ng to the epidemic, the US Fish
& Wildlife Service (USFWS) has limited access to the caves. It
has reached the west coast, killing over 6 million bats since
2006. It affects seven species of bats, and has a morality rate of
100%. With bats being important in the ecosystem with insect
control, research is vital to the survival of many species. Many
researchers are trying to find a cure, inspiring our research.
Acknowledgements
We would like to thank all those who helped us with this project, our
advisor Dr. Westenberg, the Missouri S&T iGem team, the Biology
Department, and the OURE program for our funding.
Primer
Design PCR Mini prep Digestion Transformation
T1 T2 T2-2 λ
<Figure 1- Digested Tar gene containing
plasmid (T1 and T2)>
<Figure 2- PCR EnvZ gene (E1
and E2)>
Throughout the dura5on of our project, we struggled with
the EnvZ gene not coopera5ng, making it our biggest
challenge. We received weak results from the EnvZ gene
including our PCR plasmid (Figure 2). The addi5on of the
Tar primer was successfully added to the J1101 plasmid
(Figure 1). However, the addi5on of the EnvZ primer failed
on every aPempt. Looking back, this can poten5ally be
fixed via manipula5on of the annealing temperature or by
altering the MgCl₂ concentra5on. The hypothesized
annealing temperature should be anywhere from 68०C to
65०C, despite the appearance of the primer breaking
down around 60०C.
Once the EnvZ gene is successfully added to a J1101
plasmid, the next step is to use restric5on digests on both
plasmids and get the primers into one plasmid. When the
bacterium has all the primers needed, a salt gradient will
be used and the culture should travel across the salt
gradient, which indicates that it can sense osmolality and
will travel to the change.
Poten5al Applica5ons
Our research predicts that the bacterial design could be
used in the defense against WNS. Using synthe5c biology,
researchers can add genes specific to their goal, while
using our host as a sensor and transport.