Saturation Transposon mutagenesis of Chlamydia trachomatis (360G-Wellcome-202755_Z_16_Z)

Our knowledge of the genetic basis of Chlamydia trachomatis infection and its intracellular developmental cycle has been severely hampered by the lack of genetic tools. Recently, we developed the means to introduce foreign DNA into C. trachomatis. We propose to build on this work and develop transposon technology (TraDIS) to make knock-out mutants covering the C. trachomatis genome and establish a single-gene knock-out library. Our hypothesis is that C. trachomatis retains a core of essential genes that are required for intracellular growth and development. In this project we aim to: (1) develop a delivery plasmid with an inducible transposon and show proof-of-principle that transposons operate in C. trachomatis (2) make a random, transposon-based gene knock-out library of C. trachomatis, targeted to inessential genes (3) characterise the inactivated genes by sequence analysis and make the library of mutants available. The beauty of having an inducible system is its versatility, and a TraDIS library once constructed can be used to test any number of conditions. TraDIS is a powerful technology that can be used to generate comparative genetic profiles for different conditions/treatments of infected cells during the developmental cycle. Thus, it allows key genetic events to be characterised and identified.