Date of Completion

2023

Document Type

Thesis

Degree Name

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Keywords

Amyloid Proteins, Bacillus subtilis, Bioprinting

Abstract

Biofilms are inherently rigid structures that some bacteria possess as response to harsh environmental conditions. These biofilms have components called Extracellular Polymeric Substances (EPS), which are responsible for the rigidity of biofilm structures and are beneficial in the development of bioinks. The EPS contains protein components called amyloids which are particularly attractive prospects because genetic engineering allows customizable characteristics in these structures. Bacillus subtillis have a biofilm that exhibits these ideal properties in a prospective biomaterial owed to their highly proteinaceous EPS. These EPS structures in Bacillus subtilis species are rich in TasA and other hydrophobic proteins, which greatly contribute to its mechanical stability and high surface adhesion. This biomaterial is then valuable in bioprinting technology, since there is a need for bioinks that are inherently rigid, biocompatible, and have a controllable cell behavior. Hence, by isolating the EPS and analyzing the proteinaceous component of the biomaterial, the performance of the EPS-based bioink is evaluated which can

then be a viable option for the technology. Since the simplest and widely used method of bioprinting technology is through Extrusion-Based Bioprinting, EPS-hydrogel bioinks are the most appropriate for this method. To produce the intended biofilms, liquid Bacillus subtilis cultures were prepared in an enriched media which was then used for further testing. These cultures were then screened with a crystal-violet assay to assess the viability of the strain in producing the adequate amount of biofilm. The wells that had an absorbance greater than 0.4 in the assay were reinoculated and used as a reservoir for the bio-ink in the whole study. A Bradford assay was then performed using the Bovine Serum Albumin (BSA) standard to quantify the proteins from the biofilm, averaging 937.016 μg/mL per well in the microplate. Following this, the bioink was then prepared by decellularizing the biofilm and combining it with lab-grade 5% gelatin. These then were loaded in the modified bioprinter, and printing was performed resulting to the rigid geometrical constructs. With the successful development of the bioink, prospective follow-up studies can be performed to fine-tune the process and potentially upscale the production of it for various research and clinical applications.

First Advisor

April O. Bermejo-Ambita

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