Date of Award

Summer 8-2022

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

Program/Concentration

Chemistry

Committee Director

Lesley H. Greene

Committee Member

Alvin A. Holder

Committee Member

James W. Lee

Committee Member

Margaret Mulholland

Abstract

Cyanobacteria, also known as blue-green algae, are an ancient group of microorganisms that use simple materials, such as sunlight, carbon dioxide and water, to produce energy while providing oxygen to the atmosphere by performing photosynthesis. Synthetic biology approaches have been employed with cyanobacteria as a platform to produce a range of products, such as biofuels, by inserting a series of genes into the cyanobacterial genome that will allow the conversion of metabolic intermediates to such desired products. Although these methods are promising, it is important to understand any potential bio-risk they pose. This research evaluates the potential bio-risk of genetically engineered thermophilic Thermosynechococcus elongatus BP1 (BP1) by investigating key areas of concern regarding genetically engineered microorganisms.

The first aim of this research study assesses the ability for horizontal gene transfer from engineered BP1 to wild-type bacteria, which is a major route of gene movement between microorganisms in nature. This was accomplished by co-culturing the engineered BP1 with E. coli DH5α, screening them by plating on solid media with antibiotics and verifying transfer of the transgene cassette by PCR. The second research aim characterizes the growth and survivability of engineered BP1 in varying temperatures and non-continuous lighting by monitoring the optical density over a 4-week period and calculating the colony-forming units (CFUs) during the cold and warm seasons. The final aim investigates the fate and stability of the transgenes within the genome of BP1. This was achieved by monitoring the presence and expression of the kan resistance gene monthly for 24 months using molecular biology techniques, such as PCR and RT-PCR. Illumina-based whole-genome sequencing and comparative analysis of the one-year sample was also performed to determine if any mutations were acquired within the transgene cassette. Major findings include identifying that cyanobacteria can horizontally transfer transgenes to bacteria, they do not survive well outside laboratory conditions and the transgenes within the genome remain stable for years. This body of work provides valuable insight and practical knowledge for the biotechnology community and policy makers to safely regulate the use of genetically engineered organisms for research and commercial production.

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DOI

10.25777/m4st-ve82

ISBN

9798351481340

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