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postgraduate thesis: Study of recombineering technology in Salmonella and its applications

TitleStudy of recombineering technology in Salmonella and its applications
Authors
Issue Date2012
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Yu, B. [于斌]. (2012). Study of recombineering technology in Salmonella and its applications. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4807971
AbstractIn the past few years, in vivo recombination technologies have emerged to improve the efficiency and simplicity of genetic engineering in Escherichia coli, Salmonella enterica serovar, and other gram-negative bacteria. Phage λ Red homologous recombination system is used to mediate the accurate replacement of target DNA with PCR-generated ?targeting cassettes? that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Salmonella is far lower than that in Escherichia coli. In this study, I firstly improved the recombineering-based strategy by using linear DNA targeting cassettes that contain long flanking ?arms? of sequence (ca. 1,000 base pairs) homologous to the chromosomal target. This reliable and efficient method enables multiple gene targeting procedures to be performed on a single Salmonella enterica serovar typhi Ty21a (Ty21a) chromosome in a straightforward, sequential manner with high efficiency. Secondly, I applied this improved strategy in construction of Salmonella to be live attenuated oral vaccine and tumor targeting vector. In the first part of this thesis, I describe an improved method in Ty21a. Using this strategy, I inserted three different influenza antigen expression cassettes as well as a green fluorescent protein reporter gene into four different loci on the Ty21a chromosome with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. The immune response of this vaccine was also evaluated by ELISA and ELIspots. In the second part of this thesisi, I use this improved recombineering strategy to engineer bacteria of Salmonella typhimurium (S. typhimurium) as therapeutic agents against solid tumor. In current study, a major challenge for bacterial therapy of cancer is avoiding damage to normal tissues. Consequently the virulence of bacteria must be adequately attenuated for therapeutic use. An alternative approach was developed here. By placing an essential gene under a hypoxia conditioned promoter, S. typhimurium strain SL7207 was engineered to generate strain YB1 that survives only in anaerobic conditions without otherwise affecting its functions. In breast and liver tumor bearing mice models, YB1 grew within tumor, retarding its growth, while being rapidly eliminated from normal tissues. Mice treated with SL7207 were killed by infection within short period time. Inhibition of tumor growth by YB1 was significant and was enhanced by the addition of 5-FU in breast cancer model. The development of an “obligate” anaerobic Salmonella provides a much safer bacterial vector for further development of anti-tumor therapies without compromising the other functions or tumor fitness of the bacterium as attenuation methods normally do. In summary, I have developed an efficient, robust and versatile method in genome-wide Salmonella genetic manipulation. Furthermore, I used this method to construct a recombinant Ty21a antigen-expressing vaccine strain and a tumor targeting YB1 strain.
DegreeDoctor of Philosophy
SubjectSalmonella - Genetics
Genetic recombination
Dept/ProgramBiochemistry
Persistent Identifierhttp://hdl.handle.net/10722/211555

 

DC FieldValueLanguage
dc.contributor.authorYu, Bin-
dc.contributor.author于斌-
dc.date.accessioned2015-07-17T23:11:07Z-
dc.date.available2015-07-17T23:11:07Z-
dc.date.issued2012-
dc.identifier.citationYu, B. [于斌]. (2012). Study of recombineering technology in Salmonella and its applications. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4807971-
dc.identifier.urihttp://hdl.handle.net/10722/211555-
dc.description.abstractIn the past few years, in vivo recombination technologies have emerged to improve the efficiency and simplicity of genetic engineering in Escherichia coli, Salmonella enterica serovar, and other gram-negative bacteria. Phage λ Red homologous recombination system is used to mediate the accurate replacement of target DNA with PCR-generated ?targeting cassettes? that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Salmonella is far lower than that in Escherichia coli. In this study, I firstly improved the recombineering-based strategy by using linear DNA targeting cassettes that contain long flanking ?arms? of sequence (ca. 1,000 base pairs) homologous to the chromosomal target. This reliable and efficient method enables multiple gene targeting procedures to be performed on a single Salmonella enterica serovar typhi Ty21a (Ty21a) chromosome in a straightforward, sequential manner with high efficiency. Secondly, I applied this improved strategy in construction of Salmonella to be live attenuated oral vaccine and tumor targeting vector. In the first part of this thesis, I describe an improved method in Ty21a. Using this strategy, I inserted three different influenza antigen expression cassettes as well as a green fluorescent protein reporter gene into four different loci on the Ty21a chromosome with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. The immune response of this vaccine was also evaluated by ELISA and ELIspots. In the second part of this thesisi, I use this improved recombineering strategy to engineer bacteria of Salmonella typhimurium (S. typhimurium) as therapeutic agents against solid tumor. In current study, a major challenge for bacterial therapy of cancer is avoiding damage to normal tissues. Consequently the virulence of bacteria must be adequately attenuated for therapeutic use. An alternative approach was developed here. By placing an essential gene under a hypoxia conditioned promoter, S. typhimurium strain SL7207 was engineered to generate strain YB1 that survives only in anaerobic conditions without otherwise affecting its functions. In breast and liver tumor bearing mice models, YB1 grew within tumor, retarding its growth, while being rapidly eliminated from normal tissues. Mice treated with SL7207 were killed by infection within short period time. Inhibition of tumor growth by YB1 was significant and was enhanced by the addition of 5-FU in breast cancer model. The development of an “obligate” anaerobic Salmonella provides a much safer bacterial vector for further development of anti-tumor therapies without compromising the other functions or tumor fitness of the bacterium as attenuation methods normally do. In summary, I have developed an efficient, robust and versatile method in genome-wide Salmonella genetic manipulation. Furthermore, I used this method to construct a recombinant Ty21a antigen-expressing vaccine strain and a tumor targeting YB1 strain.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.lcshSalmonella - Genetics-
dc.subject.lcshGenetic recombination-
dc.titleStudy of recombineering technology in Salmonella and its applications-
dc.typePG_Thesis-
dc.identifier.hkulb4807971-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineBiochemistry-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4807971-
dc.date.hkucongregation2012-

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