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論文名稱(外文):Development of a novel in vivo target cloning system for long DNA fragment in bacteria
指導教授(外文):Ching-Hao TengMasayuki Hashimoto
外文關鍵詞:in vivo cloningsuicide vectorgenomic island
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High-throughput sequencing technologies have made it possible to study bacteria through analyzing their genome sequences. By analyzing bacterial genomes, we can discover foreign genomic regions in the bacteria, and they might horizontally transfer from other bacteria, which are defined as genomic islands. The lengths of the genomic islands are often longer than 50 kb. PCR enables target cloning, however, it is beyond the limitation of PCR to amplify such a long DNA fragment. Here, we aim to develop an easy method to clone a large DNA fragment in in vivo, which is necessary to handle the genomic islands.
In bacteria, a suicide plasmid is generally used for gene replacement to delete a region from the chromosome. The suicide plasmid contains an upstream region of target region to be cloned, an antibiotic marker, and a downstream region of that. Through the double-crossing over process, the antibiotic marker originally located on the plasmid will be replaced with the region to be deleted on the chromosome via homologous recombination. The popped-out circular DNA is carrying the targeted region, but is non-replicable in the bacterial cell. Then, we send the popped-out circular DNA to E. coli by conjugation to rescue it as a plasmid containing target region. Finally, the plasmid can be maintained in E. coli for further manipulation.
For the aim, I constructed a suicide plasmid vector and an E. coli strain to maintain the plasmid. To validate this in vivo cloning method, we targeted a 40 kb genomic island SPI-2 encoding type III secretion machinery from Salmonella enterica serovar Typhimurium and a 70 kb fragment encoding non-ribosomal peptide synthase in Pseudomonas cichorii. Briefly, the suicide plasmid was integrated into target region through homologous recombination, and then, conjugation was carried out to rescue the popped out plasmid to E. coli. As the result, we obtained several E. coli colonies harboring the long targeted DNA fragment. PCR analysis showed that the rescued plasmid contains the target region. Complementation of ΔSPI-2 mutant of Salmonella strain by using the plasmid cloning SPI-2 region showed the cloned SPI-2 was functional. Taken together, the easy in vivo cloning system by using single suicide plasmid was developed which allowed to clone at least 70 kb DNA fragment.

中文摘要 I
Abstract II
誌謝 IV
Content V
Introduction 1
1.1 Cloning of a long DNA fragment 1
1.2 Replication control of RK2 plasmid 2
1.3 Suicide vector 4
1.4 Bacterial conjugation 5
1.5 Cloning targets 5
Specific aims 8
Materials and methods 9
2.1 Bacterial strains and culture condition 9
2.2 Cell culture 9
2.3 PCR for vector construction 9
2.4 Ligation 10
2.5 PCR with Taq polymerase 10
2.6 Plasmid transformation 10
2.7 Transformation with linearized DNA fragment by red recombination 11
2.8 Construction of cloning vector, pYW3 11
2.9 Bacterial conjugation 12
2.10 Gentamicin protection assay to determine bacterial survival within macrophage 12
2.11 Kado-Liu method 13
2.12 P1 phage transduction 13
2.13 Statistical analysis 14
Results 15
3.1 Construction of pYW3, a vector for capture plasmid 15
3.2 Cloning the SPI-2 region from Salmonella with the capture vector 16
3.3 Cloning the SPI-2 region from Salmonella with the new capture vector 18
3.4 Cloning the NRPS region from Pseudomonas cichorii by the new vector 19
3.5 Complementation assay with the popped-out plasmid 20
Discussion 22
Acknowledgment 25
Figures and Tables 26
Reference 51

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