This document summarizes a study on the synthetic production of antimicrobial peptides (AMPs) in E. coli. The purpose is to create an AMP production system in E. coli to test yield and purification parameters. Three AMPs were chosen - Spheniscin from penguins, WAM-1 from wallabies, and OH-CATH(3-34) from cobras. DNA containing the AMPs was inserted into a plasmid vector and transformed into E. coli cells. Two constructs containing Spheniscin and OH-CATH(3-34) were successfully produced, while the WAM-1 construct is still in progress. Future work includes optimizing purification, inducing AMP production

1. References/Acknowledgements
Antimicrobial Peptides
Purpose
AMP Construct Cloning Process
Future Work
ResultsSpheniscin WAM-1 OH-CATH(3-34)
Plan
Spheniscin is the AMP found
in the stomach lining of the
King Penguin. This particular
AMP is used to help preserve
food by preventing it from
being decayed by bacteria.
WAM-1 is an AMP found
in the pouches of the Wallaby
and is used to help the
immuno–deficient young
remain healthy in the early
stages of life.
OH-CATH(3-34) is an
AMP found in the King Cobra
and has shown to be highly
potent against several multi-
drug resistant strains of
bacteria.
Antimicrobial Peptides (AMPs) are naturally occurring
antibiotics found to be integral in the immune systems of
plants and animals around the globe. These antibiotics
show great promise in the future of both the medical
field with production of new drugs and the food industry
with the production of increased preservation methods.
Various production
methods for AMPs have been
used but have been limited
by production costs. The
integration of AMPs into
Escherichia coli has shown to
be very promising.
The computer generated, 3D structure
of LL-37. This AMP is found in the
human skin, respiratory tract, and
intestinal tract. It has high antibacterial
activity.
The purpose of this study is to ultimately create an
AMP production system in E. coli. Various tests can
then be performed on the AMPs to determine yield
and purification. Further experiments can then be
done to optimize these two parameters.
• Purify the DNA containing the specific AMPs
chosen for the experiments.
• Insert the DNA into the chosen plasmid vector,
PSBIC3, through DNA ligation
• Transform the plasmid into competent E. coli cells.
• Purify cells containing the DNA of interest using
CTAB
• Run a gel electrophoresis to determine the
successful cloning.
• Repeat process, adding pieces to the construct
until reaching desired result.
Constructs were first
designed with several
components such as a
promoter, a ’10XHis’ tag
for purification
purposes, a green color
fluorescing protein for
detection, and the
chosen AMP.
After designing the
constructs, the DNA
was extracted from
electrophoresis gel
slices obtained from
prior cloning cycles, or
from stock DNA. It was
then purified using a
DNA purification kit.
Once we connected our
DNA pieces together, we used
an electroporator to insert the
constructs into our competent
E. coli cells. The E. coli was
then plated onto petri dishes
and left to grow colonies of
cells overnight.
Synthetic Production of Antimicrobial Peptides in Escherichia coli
Ben Peterson
Department of Chemical Engineering, New Mexico State University, Las Cruces, New Mexico 88003
blpeters@nmsu.edu
Dr. Charles Miller
Department of Biological Engineering, Utah State University, Logan, Utah 84322
After transformation, we
purified the plasmid DNA
from the E. coli cells and cut
it with specific enzymes. We
then ran gel electrophoresis
to determine the presence
of the appropriate DNA
sequence.
• Optimization of HIS-Tag purification system using
nickel columns.
• Induce AMP production in E. coli and conduct
growth studies to determine the AMP’s effect on
cell growth.
• Conduct activity studies on several bacterial
species to determine AMP effectiveness.
• Optimize production yields of active AMPs.
Andreu, D., Rivas, L. (1998). Animal antimicrobial peptides: An
overview. Peptide Science, 47(6), 415-433.
I would like to thank Dallin, Alex, and Chad for allowing me to help in
their research. I would also like to thank Dr. Miller for letting me work in
his lab for the summer and to gain invaluable experience to be used in
the future. Lastly I would like to thank Utah State University’s
department of Biological Engineering for allowing me to come and gain
research experience and to enjoy the campus for the Summer.
Two full Antimicrobial Peptide
constructs were completed in the
time spent in the lab. These
constructs consisted of
PHTGFPEPSOhCathB0015 and
PHTGFPEPSSphenB0015. The
construct containing WAM-1 is
currently still in the cloning process. Lanes of note (left to right): DNA
ladder (lane 1),
PHTGFPEPSSphenB0015 (lane 5),
and PHTGFPEPSOhCathB0015
(lane 6)
A growth study was
conducted on the
successful constructs
without inducing AMP
production to get a growth
baseline for future
comparison.
Graph of the change in Optical Density over time
Construct Design DNA Extraction
Appropriate DNA was ligated
together by Standard Assembly
using DNA ligase and incubating
overnight.
Ligation
Transformation Gel Electrophoresis
0
0.5
1
1.5
0 2 4 6
O.D.
Hours
PHTGFPEPS
PHTGFPEPSSphenB0015
PHTGFPEPSOhCathB0015
PHTGFP