The accuracy with which the FRET signals from differently located secondary fluorophores can be detected, will be measured under a variety of conditions (e.g. temperature variation, buffer variation etc.).  The project will then move on to use other substrates supplied by participant 2, which have the primary fluorophore located in the region of the expanding loop produced during translocation.  Finally, single molecule studies will be initiated to measure the FRET signal in small volumes using confocal microfluorescence and other techniques. 

Outcomes:

Ports prepared a 2kbp PCR fragment with a fluorophore distal to a sR124 binding site.  This material was analysed using gel technology for time-resolved FRET.  The material was made available to Parma.

A series of DNA amplicons of different lengths (from 295 to 1890 bp) labelled with ATTO630 have been prepared. Measurements with them is under way.