Title Page
Contents
LIST of ABBREVIATIONS 14
ABSTRACT 16
BACKGROUND 20
1. Genetically Modified Organisms (GMOs) 20
2. Environment implications of LMM 21
3. GMM monitoring and recognition 22
1) Proteins-based method for GMO detection. 22
2) DNA-based GMO detection methods 23
4. Comparison of protein- and DNA-based GMO detection methods 25
5. PMA 26
6. Experimental technical proposal 26
7. Purpose 27
8. References 28
CHAPTER I. A novel cell-direct PCR-based method to monitor LM E. coli and C. glutamicum 37
Abstract 38
1. Introduction 39
2. Materials and Methods 40
2.1. Bacterial strains culture and cell suspension preparation 40
2.2. Determination of colony forming unit (CFU) using qPCR and plate counting 40
2.3. Design for PCR assay 41
2.4. PCR analysis 41
3. Results 42
3.1. OD600 and CFU performance[이미지참조] 42
3.2. Primer specificity and sensitivity of E. coli (AmpR) 42
3.3. Primer specificity and sensitivity of E. coli (Km2R) 43
3.4. Primer specificity and sensitivity of KmR in E. coli and C. glutamicum 43
3.5. Primer specificity and sensitivity of CmR in E. coli and C. glutamicum 44
4. Discussion 45
5. Conclusions 47
6. References 48
CHAPTER II. LM E. coli and C. glutamicum DNA standard curves in quantitative real-time PCR 62
Abstract 63
1. Introduction 64
2. Materials and Methods 65
2.1. Bacterial strains culture and cell suspension preparation 65
2.2. Design for TaqMan real-time PCR (qPCR) assay 65
2.3. DNA standard curve production 66
2.4. Dual-plex qPCR analysis 67
2.5. Statistical analysis 68
3. Results 69
3.1. qPCR parameters of AmpR for an E. coli strain with plasmid insertion 69
3.2. qPCR parameters of Km2R for an E. coli strain with genome insertion 69
3.3. qPCR parameters of KmR for E. coli and C. glutamicum strains with plasmid insertions 69
3.4. qPCR parameters of CmR for E. coli and C. glutamicum strains with plasmid insertions 70
4. Discussion 71
5. References 74
CHAPTER III. Development and application of a novel dual-plex qPCR method to quantify the LM E. coli and C. glutamicum 85
Abstract 86
1. Introduction 87
2. Materials and Methods 90
2.1. Bacterial strains culture and cell suspension preparation 90
2.2. Design of TaqMan real-time PCR (qPCR) 90
2.3. Dual-plex qPCR 90
2.4. PMA treatment for viable cell quantification 91
2.5. Determination of colony forming unit (CFU) using qPCR and plate counting 91
2.6. Statistical analysis 91
3. Results 92
3.1. Matrix effects of qPCR 92
3.2. qPCR performance of LM E. coli and C. glutamicum cell suspensions 93
3.3. Plasmid copy numbers 100
3.4. Detection limit of LM cells using PMA-qPCR 101
4. Discussion 105
5. Conclusions 109
6. References 110
CHAPTER IV. Development of enzyme-linked immunosorbent assay methods for LM E. coli and C. glutamicum monitoring 125
1. Introduction 126
2. Materials and Methods 128
2.1. Bacteria culture and cell suspension preparation 128
2.2. Total protein extraction 128
2.3. ELISA analysis 128
3. Result 130
3.1. Analysis for chloramphenicol contents of LM E. coli and C. glutamicum strains by ELISA 130
3.2. Detection limit of GM E. coli and C. glutamicum cells resistant to CmR using ELISA 130
4. Discussion 131
5. Conclusions 132
6. References 133
CHAPTER V. Conclusions 139
국문 초록 142
CHAPTER I 12
Table 1. Primers targeting antibiotic genes the ampicillin resistance gene (AmpR), the kanamycin resistance gene (KmR), the chloramphenicol resistance gene (CmR) (insert in... 50
CHAPTER II 12
Table 1. Primers and probes targeting antibiotic genes the ampicillin resistance gene (AmpR), the kanamycin resistance gene (KmR), the chloramphenicol resistance gene (CmR) (insert in plasmid), the kanamycin/neomycin resistance nptII (Km2R) (insert in E. coli genome) and the taxon-specific, single copy gene,... 76
Table 2. qPCR repeatability for AmpR/dxs by using serial dilutions of plasmid DNA extracted from three LM E. coli strains. 77
Table 3. qPCR repeatability for Km2R/dxs using serial dilutions of genomic DNA extracted from the GM E. coli strain of BW25113. 78
Table 4. qPCR repeatability for KmR/dxs and KmR/dnaA by using serial dilutions of plasmid DNA extracted from LM E. coli and LM C.glutamicum Strains, respectively. 79
Table 5. qPCR repeatability for CmR/dxs and CmR/dnaA by using serial dilutions of plasmid DNA extracted from LM E. coli and LM C.glutamicum Strains, respectively. 80
CHAPTER III 12
Table 1. qPCR and PMA-qPCR performance of the target antibiotic gene, AmpR, and the taxon-specific gene, dxs on serially diluted E. coli cell suspensions. 113
Table 2. qPCR and PMA-qPCR performance of the target antibiotic gene, Km2R and the taxon-specific gene, dxs on serially diluted E. coli cell suspensions. 114
Table 3. qPCR and PMA-qPCR performance of the target antibiotic gene, KmR and the taxon-specific gene, dxs on serially diluted E. coli cell suspensions. 115
Table 4. qPCR and PMA-qPCR performance of the target antibiotic gene, KmR, and the taxon-specific gene, dnaA on serially diluted C. glutamicum cell suspensions. 116
Table 5. qPCR and PMA-qPCR performance of the target antibiotic gene, CmR and the taxon-specific gene, dxs on serially diluted E. coli cell suspensions. 117
Table 6. qPCR and PMA-qPCR performance of the target antibiotic gene, CmR, and the taxon-specific gene, dnaA on serially diluted C. glutamicum cell suspensions. 118
Table 7. Limit of detection (LOD) confirmation using replicate PMA- qPCR runs on separate dilution series of suspension cells from E. coli. 119
Table 8. Limit of detection (LOD) confirmation using replicate PMA- qPCR runs on separate dilution series of suspension cells from C. glutamicum. 120
CHAPTER IV 13
Table 1. Analysis of chloramphenicol content of two LM strains through ELISA test. 135
Table 2. Analysis of chloramphenicol content in LM E. coli and C. glutamicum through ELISA test and derivation of detection limit. 135
BACKGROUND 9
Figure 1. Schematic drawing of a complete PCR cycle. 33
Figure 2. Schematic of TaqMan (5' nucleases) assay. 34
Figure 3. PMA working principal diagram 35
Figure 4. Experimental technical proposal 36
CHAPTER I 9
Figure 1. The diagram of plasmid constructions for three E. coli strains. (A) a modified pGAL- HIR525 (6,593 bp) plasmid harboring an ampicillin-resistance gene (AmpR); (B) a modified... 51
Figure 2. The diagram of genome modification for the BW25113 E. coli strain. A modified BW25113 E. coli strain with kanamycin/neomycin resistance nptII (Km2R) was inserted into... 52
Figure 3. The diagram of plasmid constructions for two C. glutamicum strains. (A) a modified pXMJ19~1 (10,274 bp) plasmid harboring a Chloramphenicol-resistance gene (CmR); (B) a... 53
Figure 4. E. coli (AmpR) OD600 and CFU/mL of 10 replicate qPCR cell culture suspensions(A). E. coli (Km2R) OD600 and CFU/mL of 10 replicate qPCR cell culture suspensions(B). E. coli...[이미지참조] 54
Figure 5. Plate counting of four E. coli strains for CFU/mL calculations. (A) pGAL-HIR525 on AmpR-LB medium; (B) pJ281_Alcohol-TA-Alanine on KmR-LB medium; (C) pACYC184... 55
Figure 6. C. glutamicum (KmR) OD600 and CFU/mL of 10 replicate qPCR cell culture suspensions(A). C. glutamicum (CmR) OD600 and CFU/mL of 10 replicate qPCR cell culture suspensions(B).[이미지참조] 56
Figure 7. Plate counting of four C. glutamicum strains for CFU/mL calculations. (A) pXMJ19~1 (10,274 bp) on CmR-LBBHI medium; (B) pCES20~1(10,169 bp) KmR-LBBHI medium. 57
Figure 8. Serially diluted direct cell suspension PCR analysis for E. coli strains to target the ampicillin resistance gene (AmpR) and the taxon-specific genes dxs. 100 indicates cell culture...[이미지참조] 58
Figure 9. Serially diluted direct cell suspension PCR analysis for four E. coli strains targeting the kanamycin/neomycin resistance nptII (Km2R), and the taxon-specific genes dxs. 100...[이미지참조] 59
Figure 10. Serially diluted direct cell suspension PCR analysis for E. coli and C. glutamicum strains targeting the Kanamycin resistance gene (KmR), and the taxon-specific genes dxs (E.... 60
Figure 11. Serially diluted direct cell suspension PCR analysis for E. coli and C. glutamicum strains targeting the Chloramphenicol resistance gene (CmR) and the taxon-specific genes dxs... 61
CHAPTER II 10
Figure 1. Standard curves of E. coli the ampicillin resistance gene (AmpR) and the taxon- specific genes dxs produced via qPCR assays using serial dilutions of plasmid and genomic... 81
Figure 2. Standard curves of E. coli the kanamycin/neomycin resistance nptII (Km2R) inserted into Genomics and the taxon-specific genes dxs produced via qPCR assays using serial dilutions... 82
Figure 3. Standard curves of E. coli the Kanamycin resistance gene (KmR) and the taxon- specific genes dxs and C. glutamicum the Kanamycin resistance gene (KmR) and the taxon-... 83
Figure 4. Standard curves of E. coli the Chloramphenicol resistance gene (CmR) and the taxon- specific genes dxs and C. glutamicum the Chloramphenicol resistance gene (CmR) and the... 84
CHAPTER III 10
Figure 1. Dual-plex qPCR and PMA-qPCR assays targeting AmpR and dxs using serially diluted cell suspensions. (A) Dynamic ranges of qPCR and PMA-qPCR for E. coli (AmpR). (B)... 121
Figure 2. Dual-plex qPCR and PMA-qPCR assays targeting Km2R and dxs using serially diluted cell suspensions. (A) Dynamic ranges of qPCR and PMA-qPCR for E. coli (Km2R). (B)... 122
Figure 3. Dual-plex qPCR and PMA-qPCR assays targeting KmR and dxs using serially diluted cell suspensions. (A) Dynamic ranges of qPCR and PMA-qPCR for E. coli (KmR). (B)... 123
Figure 4. Dual-plex qPCR and PMA-qPCR assays targeting CmR and dxs using serially diluted cell suspensions. (A) Dynamic ranges of qPCR and PMA-qPCR for E. coli (CmR). (B)... 124
CHAPTER IV 10
Figure 1. Example of CFU verification of E. coli and C. glutamicum for ELISA analysis 137
Figure 2. ELISA analysis standard curve generation (A) and ELISA test (B) examples. 138