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研究生:謝堉文
研究生(外文):Yu-WenHsieh
論文名稱:以細菌為平台發展新的大片段DNA複製技術
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:鄧景浩橋本昌征
指導教授(外文):Ching-Hao TengMasayuki Hashimoto
學位類別:碩士
校院名稱:國立成功大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:59
中文關鍵詞:活體克隆技術自殺式質體基因島
外文關鍵詞:in vivo cloningsuicide vectorgenomic island
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隨著科技發展,現在我們可以透過高通量定序的技術定序細菌的基因體。透過比較與分析,我們可以找到一些外來的基因序列存在於細菌的基因體中,而這些序列通常是來自細菌間水平傳播,並且被定義為基因島。這些基因島的長度通常都大於五萬對鹼基序列。聚合酶連鎖反應通常被應用在研究一個目標基因,但基因島的序列長度遠超過聚合酶連鎖反應所能放大的範圍。因此,在我的研究中,我將發展一個新的系統能夠複製像基因島這般大的長片斷鹼基序列。
在細菌的研究中,我們會透過一個自殺式質體幫助我們將細菌染色體上的基因刪除。這個自殺式質體包含欲刪除目標序列的上下游片段以及一個抗生素標記。當我們將這個自殺式質體導入細菌中,透過兩次的同源重組,我們可以將目標基因置換成抗生素標記以達成刪除基因的目的。其中,在第二次的同源重組裡,一段帶有目標序列的環狀DNA會從染色體跳出。但這個帶有目標序列的環狀DNA並不能在細菌中被保存,因此會消失在細菌中。在我的研究裡,我將這段跳出的環狀DNA透過細菌接合的方式送到大腸桿菌內保存並進行其他操作。
在我的研究中,我建構一個自殺式質體和一隻能夠保存此自殺式質體的大腸桿菌。為了證明我們系統的可行性,我利用了這個系統複製了一段在沙門氏菌中長度約四萬對鹼基序列的基因島SPI-2及一段在細菌性葉斑病菌中長度為七萬對鹼基序列的非核醣體胜肽合成酵素。在實驗中,我們先將自殺式質體送入細菌中,透過第一次同源重組,這個自殺式質體會潛入細菌染色體。之後,我們利用細菌接合將發生第二次同源重組所跳出並帶有目標序列的質體送到大腸桿菌中。在實驗結果中,我們得到一些含有目標DNA質體的細菌,並透過聚合酶連鎖反應進行確認。此外,將含有基因島SPI-2的質體送入SPI-2突變的沙門氏菌能夠成功回補SPI-2的功能,這個結果顯示通過此複製技術所複製出的DNA片段是有功能的。綜合以上實驗結果,我們發展了一個新的活體大片段複製技術,且這個技術只需要一個自殺式質體且已知最少能夠用來複製長七萬對鹼基序列的DNA片段。

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


1.Hernandez, D., et al., De novo bacterial genome sequencing: millions of very short reads assembled on a desktop computer. Genome Research, 2008. 18(5): p. 802-809.
2.Che, D., M.S. Hasan, and B. Chen, Identifying pathogenicity islands in bacterial pathogenomics using computational approaches. Pathogens, 2014. 3(1): p. 36-56.
3.Dobrindt, U., et al., Genomic islands in pathogenic and environmental microorganisms. Nature Reviews Microbiology, 2004. 2(5): p. 414-424.
4.Hacker, J. and J.B. Kaper, Pathogenicity islands and the evolution of microbes. Annu Rev Microbiol, 2000. 54: p. 641-79.
5.Hoseini, S.S. and M.G. Sauer, Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering. Journal of Biological engineering, 2015. 9(1): p. 1.
6.Xia, H., et al., Construction of an ordered cosmid library of S. avermitilis for genetic modification of the industrial strains. Chin J Antibiot, 2009. 34: p. 340-343.
7.Béjà, O., et al., Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environmental Microbiology, 2000. 2(5): p. 516-529.
8.Huang, J., et al., A strategy for seamless cloning of large DNA fragments from Streptomyces. BioTechniques, 2015. 59(4): p. 193.
9.Wild, J., et al., A broad-host-range in vivo pop-out and amplification system for generating large quantities of 50-to 100-kb genomic fragments for direct DNA sequencing. Gene, 1996. 179(1): p. 181-188.
10.Kvitko, B.H., I.A. McMillan, and H.P. Schweizer, An improved method for oriT-directed cloning and functionalization of large bacterial genomic regions. Applied and Environmental Microbiology, 2013. 79(16): p. 4869-4878.
11.Gibson, D.G., et al., Creation of a bacterial cell controlled by a chemically synthesized genome. Science, 2010. 329(5987): p. 52-56.
12.Endy, D., Foundations for engineering biology. Nature, 2005. 438(7067): p. 449-453.
13.Del Solar, G., et al., Replication and control of circular bacterial plasmids. Microbiology and Molecular Biology Reviews, 1998. 62(2): p. 434-464.
14.Lin-Chao, S. and S.N. Cohen, The rate of processing and degradation of antisense RNAI regulates the replication of ColE1-type plasmids in vivo. Cell, 1991. 65(7): p. 1233-1242.
15.Tomizawa, J.-i., et al., Inhibition of ColE1 RNA primer formation by a plasmid-specified small RNA. Proceedings of the National Academy of Sciences, 1981. 78(3): p. 1421-1425.
16.Kües, U. and U. Stahl, Replication of plasmids in gram-negative bacteria. Microbiological Reviews, 1989. 53(4): p. 491-516.
17.Heuer, H. and K. Smalla, Plasmids foster diversification and adaptation of bacterial populations in soil. FEMS Microbiology Reviews, 2012. 36(6): p. 1083-1104.
18.Lowbury, E., et al., Sensitivity of Pseudomonas aeruginosa to antibiotics: emergence of strains highly resistant to carbenicillin. The Lancet, 1969. 294(7618): p. 448-452.
19.Kostyal, D.A., et al., Replication of an RK2 miniplasmid derivative in vitro by a DNA/membrane complex extracted from Escherichia coli: involvement of the dnaA but not dnaK host proteins and association of these and plasmid-encoded proteins with the inner membrane. Plasmid, 1989. 21(3): p. 226-237.
20.Figurski, D.H., et al., Broad host range plasmid RK2 encodes multiple kil genes potentially lethal to Escherichia coli host cells. Proceedings of the National Academy of Sciences, 1982. 79(6): p. 1935-1939.
21.Ayres, E.K., et al., Precise deletions in large bacterial genomes by vector-mediated excision (VEX): the trfA gene of promiscuous plasmid RK2 is essential for replication in several gram-negative hosts. Journal of Molecular Biology, 1993. 230(1): p. 174-185.
22.Santos, P.M., et al., New broad-host-range promoter probe vectors based on the plasmid RK2 replicon. FEMS microbiology letters, 2001. 195(1): p. 91-96.
23.Wilson, J.W., D.H. Figurski, and C.A. Nickerson, VEX-capture: a new technique that allows in vivo excision, cloning, and broad-host-range transfer of large bacterial genomic DNA segments. Journal of Microbiological Methods, 2004. 57(3): p. 297-308.
24.Kovach, M.E., et al., Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene, 1995. 166(1): p. 175-176.
25.Blatny, J.M., et al., Improved broad-host-range RK2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. Plasmid, 1997. 38(1): p. 35-51.
26.Philippe, N., et al., Improvement of pCVD442, a suicide plasmid for gene allele exchange in bacteria. Plasmid, 2004. 51(3): p. 246-255.
27.Ortiz-Martín, I., et al., Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria. Journal of Microbiological Methods, 2006. 67(3): p. 395-407.
28.Dean, D., A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids. Gene, 1981. 15(1): p. 99-102.
29.Gay, P., et al., Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. Journal of Bacteriology, 1985. 164(2): p. 918-921.
30.Maloy, S.R. and W.D. Nunn, Selection for loss of tetracycline resistance by Escherichia coli. Journal of Bacteriology, 1981. 145(2): p. 1110.
31.Blomfield, I., et al., Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature‐sensitive pSC101 replicon. Molecular Microbiology, 1991. 5(6): p. 1447-1457.
32.Ried, J.L. and A. Collmer, An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis. Gene, 1987. 57(2): p. 239-246.
33.Selvaraj, G. and V. Iyer, Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. Journal of Bacteriology, 1983. 156(3): p. 1292-1300.
34.Davison, J., Genetic exchange between bacteria in the environment. Plasmid, 1999. 42(2): p. 73-91.
35.Lanka, E. and B.M. Wilkins, DNA processing reactions in bacterial conjugation. Annual Review of Biochemistry, 1995. 64(1): p. 141-169.
36.Ilangovan, A., S. Connery, and G. Waksman, Structural biology of the Gram-negative bacterial conjugation systems. Trends in Microbiology, 2015. 23(5): p. 301-310.
37.Derbyshire, K.M. and N.S. Willetts, Mobilization of the non-conjugative plasmid RSF1010: a genetic analysis of its origin of transfer. Molecular and General Genetics MGG, 1987. 206(1): p. 154-160.
38.Bale, M., M. Day, and J. Fry, Novel method for studying plasmid transfer in undisturbed river epilithon. Applied and Environmental Microbiology, 1988. 54(11): p. 2756-2758.
39.Hill, K., A. Weightman, and J. Fry, Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10. Applied and Environmental Microbiology, 1992. 58(4): p. 1292-1300.
40.Majowicz, S.E., et al., The global burden of nontyphoidal Salmonella gastroenteritis. Clinical Infectious Diseases, 2010. 50(6): p. 882-889.
41.McClelland, M., et al., Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature, 2001. 413(6858): p. 852-856.
42.Sabbagh, S.C., et al., So similar, yet so different: uncovering distinctive features in the genomes of Salmonella enterica serovars Typhimurium and Typhi. FEMS Microbiology Letters, 2010. 305(1): p. 1-13.
43.Figueira, R. and D.W. Holden, Functions of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system effectors. Microbiology, 2012. 158(5): p. 1147-1161.
44.Wang, X., et al., Genomic and transcriptomic analysis of the endophytic fungus Pestalotiopsis fici reveals its lifestyle and high potential for synthesis of natural products. BMC genomics, 2015. 16(1): p. 1.
45.Gross, H. and J.E. Loper, Genomics of secondary metabolite production by Pseudomonas spp. Natural Product Reports, 2009. 26(11): p. 1408-1446.
46.Walsh, C.T., The chemical versatility of natural-product assembly lines. Accounts of Chemical Research, 2007. 41(1): p. 4-10.
47.Donia, M.S., et al., A systematic analysis of biosynthetic gene clusters in the human microbiome reveals a common family of antibiotics. Cell, 2014. 158(6): p. 1402-1414.
48.Cane, D.E. and C.T. Walsh, The parallel and convergent universes of polyketide synthases and nonribosomal peptide synthetases. Chemistry & Biology, 1999. 6(12): p. R319-R325.
49.Bachmann, B.O. and J. Ravel, Methods for in silico prediction of microbial polyketide and nonribosomal peptide biosynthetic pathways from DNA sequence data. Methods in Enzymology, 2009. 458: p. 181-217.
50.Nguyen, K.T., et al., Combinatorial biosynthesis of novel antibiotics related to daptomycin. Proceedings of the National Academy of Sciences, 2006. 103(46): p. 17462-17467.
51.Bradbury, J.F., Guide to plant pathogenic bacteria. 1986: CAB International.
52.Pauwelyn, E., et al., New linear lipopeptides produced by Pseudomonas cichorii SF1-54 are involved in virulence, swarming motility, and biofilm formation. Molecular Plant-Microbe Interactions, 2013. 26(5): p. 585-598.
53.Huang, C.-J., et al., Characterization of cichopeptins, new phytotoxic cyclic lipodepsipeptides produced by Pseudomonas cichorii SF1-54 and their role in bacterial midrib rot disease of lettuce. Molecular Plant-Microbe Interactions, 2015. 28(9): p. 1009-1022.
54.Baba, T., et al., Construction of Escherichia coli K‐12 in‐frame, single‐gene knockout mutants: the Keio collection. Molecular Systems Biology, 2006. 2(1).
55.Kolatka, K., et al., Replication and partitioning of the broad-host-range plasmid RK2. Plasmid, 2010. 64(3): p. 119-134.
56.Buchmeier, N.A. and F. Heffron, Intracellular survival of wild-type Salmonella typhimurium and macrophage-sensitive mutants in diverse populations of macrophages. Infection and Immunity, 1989. 57(1): p. 1-7.
57.Cirillo, D.M., et al., Macrophage‐dependent induction of the Salmonella pathogenicity island 2 type III secretion system and its role in intracellular survival. Molecular Microbiology, 1998. 30(1): p. 175-188.
58.Poh, J., et al., SteC is a Salmonella kinase required for SPI‐2‐dependent F‐actin remodelling. Cellular Microbiology, 2008. 10(1): p. 20-30.
59.Meresse, S., et al., Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella. Cellular Microbiology, 2001. 3(8): p. 567-577.
60.Miao, E.A., et al., Salmonella effectors translocated across the vacuolar membrane interact with the actin cytoskeleton. Molecular Microbiology, 2003. 48(2): p. 401-415.
61.Richer, E., et al., N-ethyl-N-nitrosourea–induced mutation in ubiquitin-specific peptidase 18 causes hyperactivation of IFN-αβ signaling and suppresses STAT4-induced IFN-γ production, resulting in increased susceptibility to Salmonella typhimurium. The Journal of Immunology, 2010. 185(6): p. 3593-3601.
62.Birren, B., V. Mancino, and H. Shizuya, Bacterial artificial chromosomes. Genome analysis: A Laboratory Manual, 1999. 3: p. 241-245.
63.Figurski, D.H. and D.R. Helinski, Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proceedings of the National Academy of Sciences, 1979. 76(4): p. 1648-1652.
64.Hershfield, V., et al., Plasmid ColE1 as a molecular vehicle for cloning and amplification of DNA. Proceedings of the National Academy of Sciences, 1974. 71(9): p. 3455-3459.
65.Shafferman, A. and D. Helinski, Structural properties of the beta origin of replication of plasmid R6K. Journal of Biological Chemistry, 1983. 258(7): p. 4083-4090.
66.Tao, Q. and H.B. Zhang, Cloning and stable maintenance of DNA fragments over 300 kb in Escherichia coli with conventional plasmid-based vectors. Nucleic Acids Research, 1998. 26(21): p. 4901-4909.
67.Yamaguchi, Y., J.-H. Park, and M. Inouye, Toxin-antitoxin systems in bacteria and archaea. Annual Review of Genetics, 2011. 45: p. 61-79.
68.Chen, Y. and H.P. Erickson, In Vitro Assembly Studies of FtsZ/Tubulin-like Proteins (TubZ) from Bacillus Plasmids EVIDENCE FOR A CAPPING MECHANISM. Journal of Biological Chemistry, 2008. 283(13): p. 8102-8109.
69.Hwang, L.C., et al., ParA‐mediated plasmid partition driven by protein pattern self‐organization. The EMBO journal, 2013. 32(9): p. 1238-1249.
70.Gallo, A., et al., Identification and characterization of the polyketide synthase involved in ochratoxin A biosynthesis in Aspergillus carbonarius. International Journal of Food Microbiology, 2014. 179: p. 10-17.
71.Zhang, H., A. Rokas, and J.C. Slot, Two different secondary metabolism gene clusters occupied the same ancestral locus in fungal dermatophytes of the Arthrodermataceae. PloS one, 2012. 7(7): p. e41903.
72.Baltz, R.H., Genomics and the ancient origins of the daptomycin biosynthetic gene cluster. The Journal of Antibiotics, 2010. 63(8): p. 506-511.
73.Blattner, F.R., et al., The complete genome sequence of Escherichia coli K-12. science, 1997. 277(5331): p. 1453-1462.
74.Simon, R., U. Priefer, and A. Pühler, A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Nature Biotechnology, 1983. 1(9): p. 784-791.
75.Boyer, H.W. and D. Roulland-dussoix, A complementation analysis of the restriction and modification of DNA in Escherichia coli. Journal of Molecular Biology, 1969. 41(3): p. 459-472.


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