A method for generating precise gene deletions and insertions in Escherichia coli.

A simple and general method for disrupting chromosomal genes and introducing insertions is described. This procedure involves eliminating wild-type bacterial genes and introducing mutant alleles or other insertions at the original locus of the wild-type gene. To demonstrate the utility of this approach, the tig gene of Escherichia coli was replaced by homologous recombination with a cassette containing the chloramphenicol resistance gene and the sacB gene.

Trigger factor assisted folding of green fluorescent protein.

Guanidine induced equilibrium and kinetic folding of a variant of green fluorescent protein (F99S/M153T/V163A, GFPuv) was studied. Using manual mixing and stopped-flow techniques, we combined different probes, including tryptophan fluorescence, chromophore fluorescence and reactivity with DTNB, to trace the spontaneous and TF-assisted folding of guanidine denatured GFPuv. We found that both unfolding and refolding of GFPuv occurred in a stepwise manner and a stable intermediate was populated under equilibrium conditions.