Title page for ETD etd-04292011-133956


Type of Document Master's Thesis
Author Riggs, Sarah Danielle
Author's Email Address srigg004@vt.edu
URN etd-04292011-133956
Title Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria
Degree Master of Science
Department Biological Sciences
Advisory Committee
Advisor Name Title
Yang, Zhaomin Committee Chair
Falkinham, Joseph O. III Committee Co-Chair
Popham, David L. Committee Member
Keywords
  • transposon
  • Mycobacterium smegmatis
  • Tuberculosis
  • TetR
  • essential genes
  • genetics
Date of Defense 2011-04-18
Availability unrestricted
Abstract
The continuing emergence of multi-drug resistant Mycobacterium tuberculosis is threatening the ability to treat tuberculosis (TB) worldwide. The development of new anti-TB drugs requires new approaches and new drug targets. In this study, a mariner-based transposon, TnQuoVadis, was constructed to identify essential genes as potential drug targets. This transposon has an outward-facing anhydrotetracycline (ATc)-inducible promoter at each end. A mutant with TnQuoVadis inserted upstream of an essential gene may display normal growth in the presence of ATc, but exhibit no growth or severely diminished growth in the absence of ATc. TnQuoVadis was placed onto a vector with a temperature sensitive replication origin for more efficient mutagenesis of mycobacteria. In a preliminary genetic screen using the model organism Mycobacterium smegmatis, 13 mutants with ATc-dependent growth were identified. Identification of the insertion sites by cloning and sequencing indicated that there were nine genetic loci containing transposon insertions upstream of essential gene candidates in M. smegmatis. Further analysis of these genes indicated that many were previously known essential in both M. smegmatis and M. tuberculosis. These results demonstrate that TnQuoVadis and its delivery system can be utilized for the identification of essential genes in mycobacteria
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