Date of Award:

8-2023

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biology

Committee Chair(s)

Justin A. Jones

Committee

Justin A. Jones

Committee

Randolph V. Lewis

Committee

Ryan Jackson

Abstract

Overview: People in India and China have produced silk textiles for thousands of years. Silk is a biodegradable, biocompatible compound used in the production of clothing, bedding, furniture, industrial materials, and medical applications. Over the last 30 years, research has increasingly investigated silk’s antimicrobial effects and how to augment its natural abilities. Antimicrobial peptides, or AMPs, are also an area of increasing interest as the rise of antibiotic resistance reduces the efficacy of current treatments. This project plans to systematically synthesize a fusion protein that incorporates the beneficial properties of each constituent into commercial silkworms.

Innovation: This project seeks to introduce the AMP LL-37 into silkworm silk through genetic engineering. This will allow for the production of antimicrobial silk in a commercially viable and well-established industrial insect with little to no modification required for current industrial operations after initial development while not requiring post-treatments to the fibers. Integration into the silk protein will also allow for greater antimicrobial efficacy over an extended period. In addition, it will not wash away as quickly as current antimicrobial treatments that are coatings or sprays.

Brief Process Summary: LL-37 was chosen based on protein size, cost, mechanism of action, and amino acids readily available in the natural silkworm diet. Next, the construct design and synthesis via cloning in E. coli in occurred in preparation for the CRISPR/Cas9 system. Using the LC-NHEJ pBsK expression system, the LL-37 sequence, and proline linker were cloned into the CRISPR/Cas9 expression vector in Escherichia coli. Upscaling of E. coli culture to produce enough DNA for electroporation into the silkworm line was then performed. Then, silkworm eggs were transformed via electroporation and a specialized protocol. Next, silkworm rearing took approximately two months. This was followed by visualization of the insert into the silk through a GFP tag and via a dot blot antibody test. Mechanical data on the silk fibers, including fiber diameter, tensile strength, and the energy to break, were also collected. The final steps included determining the antimicrobial activity via a bacterial viability stain using fluorescence microscopy to quantify the level of effectiveness.

Medical Relevance: Antimicrobial peptides are increasingly in demand as an alternative to antibiotic treatments due to antibiotic resistance. Antimicrobial peptides, including LL37, are known to have efficacy against common strains of bacteria found on the human body, including Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus aureus (the organism responsible for potential MRSA infections). In addition, having a readily available silk fiber has benefits in the production of clean medical sheets and bedding to reduce the chance of contamination, increasing hygiene in exercise clothing, and reducing the risk of surgical site suture infections, therefore reducing the need for antibiotic treatments.

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