Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering.

The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways.

Knowledge of aging and attitudes toward older people. A survey of Australian podiatric medical students.

Podiatric medical students in Australia were surveyed to evaluate their reasons for entering podiatric medicine, knowledge of aging, attitudes toward older people, perceptions of treatment efficacy, and desire to specialize in geriatrics. Few students plan to specialize in geriatrics upon graduation (4%), with most preferring general practice (25%) or sports medicine (21%). However, knowledge of aging was good, and students had favorable attitudes toward older people and considered treatment of older people to be effective. Few age- or gender-related effects were observed. It is concluded that students' lack of desire to specialize in geriatrics may be due not to unfavorable perceptions of older people but rather to the low profile and limited development of geriatrics as a specialty area within the podiatric medical profession.