臺灣博碩士論文加值系統

聚羥基丁酸酯 (P3HB)由於其生物可降解性及生物相容性使它獲得了許多關注，先前我們成功將嗜熱菌C. manganoxidans的P3HB合成基因phaCAB轉殖到E. coli中表達，然而過程中仍需添加抗生素及誘導劑。為了能夠於不添加抗生素及誘導劑的情況下生產P3HB，我們構築了三個含有穩定系統的新質體，分別是含有主動分隔系統 (pUC19alp7)、toxin/antidote系統 (pUC19parB)及兩者皆具(pUC19ap)，在質體穩定性測試的實驗中，pUC19parB展現了良好的穩定性，並被用於構築表達P3HB合成蛋白的載體。構築出的質體p19BCAB1被轉殖到E. coli BL21 (DE3)中進行P3HB生產及穩定性測試實驗。在穩定性的測試實驗中顯示p19BCAB1並不如預期的穩定，此結果與P3HB生產結果一致，於含抗生素的培養基中進行發酵實驗，P3HB content、P3HB濃度及P3HB yield分別為47.76±1.29%, 5.62±0.09 g/L, 及0.22±0.02 g/g glucose，然而不添加抗生素時分別只有14.14±0.13%, 1.06±0.11 g/L, 及0.05±0.0005 g/g glucose，分別減少了81%, 70%及77%，而減少的部份為丟失質體後菌株將碳流導至其他產物上，實驗結果中也看到的乙酸的累積。

Poly-3-hydroxybutyrate (P3HB) featuring of its biodegradability and biocompatibility make it gain a lot of attention. In our previous research, we successfully transferred the P3HB biosynthetic genes phaCAB from thermophilic C. manganoxidans to E. coli while antibiotics and inducers are necessary during the fermentation. In order to produce P3HB without the addition of antibiotics and inducers, we have constructed three new plasmids with stable plasmid systems, including active partitioning system (pUC19alp7), toxin/antidote system (pUC19parB), and both (pUC19ap). pUC19parB showed good stability in the experiments of plasmid stability test and was used as a vector expressing P3HB synthetic proteins. The constructed plasmid p19BCAB1 was transferred to E. coli BL21 (DE3) for P3HB production and stability test. In the stability test, p19BCAB1 was not stable as expected. This result was consistent with the P3HB production. The fermentation experiment was carried out in the antibiotic-containing medium. The P3HB content, P3HB concentration and P3HB yield were 47.76±1.29%, 5.62±0.09 g/L, and 0.22±0.02 g/g glucose respectively. However, when no antibiotics were added, only 14.14±0.13%, 1.06±0.11 g/L, and 0.05±0.0005 g/g glucose which reduced by 81%, 70% and 77%, respectively. After the plasmid loss occurs, the carbon flow is led to produce other products. The accumulation of acetate was observed in the experimental results.

Table of content
1. Introduction 1
1.1 Motivation 1
1.2 Literature review 2
1.2.1 Introduction of P3HB and PHAs 2
1.2.2 Production of P3HB by microorganisms 2
1.2.3 Plasmid stability system 3
2. Materials and methods 5
2.1 Bacteria strains and plasmid 5
2.2 Sequence and ligation-independent cloning (SLIC) 5
2.3 DNA techniques 5
2.3.1 Extraction of plasmid 5
2.3.2 DNA purification of gel extraction 6
2.3.3 Agarose gel electrophoresis 7
2.3.4 Polymerase chain reaction 7
2.3.5 Demethylation 7
2.3.6 Vector digestion 7
2.3.7 T4 DNA polymerase treatment 8
2.3.8 Transformation (Chemical transformation) 8
2.4 SDS-PAGE 8
2.5 Culture media and growth conditions 9
2.6 Plasmid stability test 9
2.7 P3HB content 10
3. Results 11
3.1 Construction of recombinant plasmids 11
3.2 Plasmid stability test of new plasmids 11
3.3 SDS-PAGE of BL21 (DE3)/p19BCAB1 12
3.4 P3HB production of shake flask experiment 13
4. Discussion 14
5. Conclusion 15
6. References 16
Appendix I. The list of chemicals 48
Appendix II. The list of instruments 49
Appendix III. Calibration curves 50

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