Extrachromosomal elements of Chlamydia: Development of an efficient transformation system for Chlamydia trachomatis. (360G-Wellcome-091296_Z_10_Z)

The inability to take a direct genetic approach to study the function and regulation of chlamydial genes has held back research progress. Whilst the possibility of introducing foreign DNA has been established the protocols are extremely inefficient, haphazard and irreproducible. The accumulated evidence strongly suggests that the replicating metabolically active form of the organism, the reticulate body (RB) is the bacterial form susceptible to DNA uptake. Accordingly we propose a systematic t echnology development programme to determine the most efficient way to introduce 'foreign' DNA firstly, to the chlamydial inclusion and secondly into chlamydia themselves (RBs). Our proposal has seven key objectives, the first three are to determine the optimal cell line, chlamydial strain and conditions for introducing DNA into the inclusion. The next steps then involve optimising the protocol for RB transformation using the phage genome (a self amplifying molecule that negates the need for sel ection) and the final step is to develop a vector carrying a selectable marker. Having established the optimal conditions and vector our sixth objective is to ensure this process is reproducible in another lab and finally we wish to disseminate this knowledge via a working protocol and workshop to our colleagues in the field.