I studied the mechanism involved in the spreading of antibiotic resistance genes by genetic manipulations in korB of RK2 plasmid.
Prof. Christopher M Thomas’ Lab (Founder of Plasmid Biology Conference; Director of the Graduate School), University of Birmingham, UK (2005-2010).
KorB is a negatively charged (-21) global regulatory protein that interacts with other regulatory proteins in a multi-resistant (Amp, Km, Tet) plasmid RK2 (60kb; 1-4 copy number) to control its partitioning to all gram negative, some gram positive, and eukaryotic cells.
My work showed for the first time that the balance of charge in the central region of KorB, rather than positively charged amino-acids alone, is responsible for its binding with DNA.
- Determined the regions of a regulatory protein KorB involved in gene silencing, gene repression and DNA binding.
- Identified amino acids in the central region of KorB responsible for interaction with other regulatory proteins (i.e. KorA and TrbA), distal and proximal repression of genes, and DNA binding.
Methods and assays
- Designed and developed 3-plasmid system in E.coli to study gene regulation and gene silencing.
- Expressed and purified 21 proteins (WT and mutants of KorB, WT KorA, WT TrbA).
- Characterized 18 proteins using biochemical and biophysical techniques.
- Developed assays to study Protein-Protein and Protein-DNA interactions.
- Techniques: DNA purification, PCR, Gene cloning and sequencing, Mutagenesis (random, deletion and substitution), Antibodies purification, Protein purification, SDS-PAGE, Western blot, DNA footprinting, Gene silencing assays, Gene repression assays, Radioactive labeling of DNA, Protein-protein interactions, Protein-DNA interactions, Protein co-purification assays, Analytical Ultracentrifugation, etc.
Thesis: Global regulation in broad host range plasmid RK2: Functional dissection of partitioning and global regulatory protein KorB.
Global regulation in broad host range plasmid RK2: Mapping the region of partitioning/global regulation protein KorB that interacts with repressors KorA and TrbA. Sidra Muntaha · Kalliopi Kostelidou · Maciej R. Lukaszewicz · Le. Bingle · Christopher M. Thomas
Distribution of the partitioning protein KorB on the genome of IncP-1 plasmid RK2. Chung-Min Chiu · Susan E Manzoor · Sarah M Batt · Sidra Muntaha · Christopher M Thomas
A single aromatic residue in transcriptional repressor protein KorA is critical for cooperativity with its co-regulator KorB. Lewis E H Bingle · Karthik V Rajasekar · Sidra Muntaha · Vinod Nadella · Christopher M Thomas
Order and Disorder in the Domain Organization of the Plasmid Partition Protein KorB. Karthik Rajasekar · Sidra Muntaha · Jeremy R H Tame · Sireesha Kommareddy · David J Scott
Conference Paper: Regulation of maintenance and transfer of promiscuous plasmids. C. M. Thomas · L. E. H. Bingle · Sidra Muntaha · K. Rajasekar · S. A. White
Data: Supplementary Material. Lewis E H Bingle · Karthik V Rajasekar · Sidra Muntaha · Vinod Nadella · Christopher M Thomas
Presentations in Conferences
- Gordon Conference: Chromosomal Dynamics, USA 2007
- Plasmid Biology Conference, USA 2006
- Society for General Microbiology meeting Fighting Infections, Bergen, Norway 2005
National conferences (UK)
- Society for General Microbiology meeting, Scotland 2008
- Biochemical Society meeting, Manchester 2008
- Mobile Genetic Elements meeting, Birmingham 2008