Honors Theses and Capstones

Date of Award

Spring 2021

Project Type

Senior Honors Thesis

College or School

CEPS

Department

Chemical Engineering

Program or Major

Bioengineering

Degree Name

Bachelor of Science

First Advisor

Wu Kang

Second Advisor

Guo Wu

Abstract

Thermophilic bacteria have attracted research interest due to their ability to grow at high temperatures ranging from 45 °C to 75 °C with some extreme thermophiles able to survive nearly boiling temperatures. They are valuable sources of thermostable biocatalysts and many have great potential as industrial hosts for biofuel production because fermentation at high temperatures has advantages such as reduced processing costs and lower risk of contamination compared with mesophilic bacteria. For example, some Geobacillus species has been shown to have high solvent tolerance, making them good candidates as host for alcohol production. To develop them as industrial hosts, large scale genetic engineering to rewire the metabolic network is necessary, which includes inducible gene expression for optimizing of the level of proteins in the network. However, most genetic tools currently available do not function in Geobacillus due to poor thermostability. In this project, we aim to develop thermostable inducible expression elements. LacI is a regulator which controls the gene expression of the lac operon in response to lactose. Many non-metabolizable lactose analogs, such as IPTG, have been developed to decouple the regulation from the metabolic network, making it the most commonly used inducible gene expression tool. Three mutants were made by introducing a point mutation to change a charge residue to a non-polar residue (K84M, K84I, K84L) to increase its thermostability. These LacI mutants were coupled with a thermostable GFP as the reporter to test its functionality in Geobacillus. These mutants are expected to also function in other thermophiles after further organism specific codon optimization.

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