GIST Repository
College of Engineering
Department of Environment and Energy Engineering
3. Theses(Master)
Development of Fluorescence-Based Reporter Systems for Acetogenic Bacterium, Eubacterium callanderi KIST612
- Abstract
- Eubacterium callanderi KIST612, an acetogen and anaerobic bacterium, is a promising biocatalyst for using C1 gases (e.g., CO and CO2) to produce valuable chemicals. Despite its whole genome and genetic toolboxes secured, establishing the reporter system is still required for better suitability and sensitivity due to limitations in some available reporter genes and strain characteristics. The objective of this study is to establish fluorescent reporter systems to effectively operate in anaerobic and CO-rich conditions. As an initial step, we investigated the intrinsic fluorescence (autofluorescence) properties of the strain to avoid high background signals. After analyzing the autofluorescence (420-440/480-530 nm) of wild-type E. callanderi KIST612 using excitation- emission matrix (EEM) data, fluorescence-activating and absorption-shifting tag (FAST) were tested. The obtained data showed that FAST could be expressed in E. callanderi KIST612 under anaerobic and CO-rich condition. To test the capability under various CO conditions, FAST was applied to CO-sensing biosensor in E. coli DH5α. This study seeks to create a comprehensive fluorescent reporter system that is applicable to E. callanderi KIST612 and could be extended to other acetogenic strains.
- Issued Date
- 2025
- Type
- Thesis
- Alternative Author(s)
- Ardiwinata Tantri
- Department
- 대학원 환경에너지공학부
- Advisor
- Chang, In Seop
- Table Of Contents
- Abstract i
Table of Contents ii
List of Tables iv
List of Figures v
I. INTRODUCTION 1
1.1. Background and Literature Review 1
1.1.1. Acetogen 1
1.1.1.1. Wood-Ljungdahl Pathway 3
1.1.1.2. Eubacterium callanderi KIST612 3
1.1.1.3. Toolbox Availability and Limitation 5
1.1.2. Fluorescence-Based Reporter System 6
1.1.2.1. Principle of Fluorescence 6
1.1.2.2. Fluorescent Protein as a Reporter Gene 8
1.1.3. Challenges on Constructing Fluorescence Reporter System in Acetogen 9
1.1.3.1. Autofluorescence 9
1.1.3.2. CO Exposure 10
1.2. Research Objectives 10
II. MATERIALS AND METHODS 12
2.1. Bacteria Strains and Culture Conditions 12
2.1.1. Escherichia coli DH5α 12
2.1.2. Eubacterium callanderi KIST612 12
2.2. Confirmation of the Autofluorescence of E. callanderi KIST612 12
2.3. Plasmid Construction and Cloning 13
2.3.1. Securing and Designing FAST1 Reporter System 13
2.3.2. Polymerase Chain Reaction (PCR) 15
2.3.3. Gibson Assembly 17
2.3.4. Confirmation and Purification 19
2.3.5. Transformation 19
2.4. Fluorescent-Activating and Absorption Shifting Tags (FAST) Assay 20
2.5. Quantitative and Qualitative Measurements 20
III. RESULTS AND DISCUSSIONS 21
3.1. Autofluorescence of E. callanderi KIST612 21
3.2. Selection of Fluorescent Reporter System Candidates 23
3.3. FAST1 Expression in E. callanderi KIST612 Under Autotrophic and Heterotrophic Conditions 27
3.4. FAST1 Application in CO Biosensor Under CO Partial Pressure Variations 29
IV. CONCLUSIONS 32
REFERENCES 33
ACKNOWLEDGEMENT 39
- Degree
- Master
- Appears in Collections:
- Department of Environment and Energy Engineering > 3. Theses(Master)
- 파일 목록
-
-
관련 파일이 존재하지 않습니다.
-
Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.