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 coliBen Peterson

Department of Chemical Engineering, New Mexico State University, Las Cruces, New Mexico 88003 blpeters@nmsu.edu

Dr. Charles MillerDepartment 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 1 2 3 4 5 600.20.40.60.8

11.21.4

PHTGFPEPSPHTGFPEPSSphenB0015PHTGFPEPSOhCathB0015PHTGFP

Hours

O.D

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