Engineering Gene Expression

Genetic modification of P. putida is highly relevant for metabolic engineering of producing strains. Modifications of interest consist of deletions and insertions attributing new features to the genome [66]. Such manipulations can be obtained by (i) homologous recombination (mediated by the RecA catalytic activity), (ii) site-specific recombination (subordinated to either the resolvase-invertase family or the FLP-fRT, Cre-loxP family, also referred to as the Int family), and 

Mini-Tn5 and TniO derivatives Gene insertion mutagenesis, promoter probing, chromosomal insertion of cloned DNA [57-60] 

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Thomas CH et al (2002) Engineering gene expression and protein synthesis by modulation of nuclear shape. Proc Natl Acad Sci USA 99(4) 1972-1977... 

The methods involved in the production of proteins in microbes are those of gene expression. Several plasmids for expression of proteins having affinity tails at the C- or N-terminus of the protein have been developed. These tails are usefiil in the isolation of recombinant proteins. Most of these vectors are commercially available along with the reagents that are necessary for protein purification. A majority of recombinant proteins that have been attempted have been produced in E. Coli (1). In most cases these recombinant proteins formed aggregates resulting in the formation of inclusion bodies. These inclusion bodies must be denatured and refolded to obtain active protein, and the affinity tails are usefiil in the purification of the protein. Some of the methods described herein involve identification of functional domains in proteins (see also Protein engineering). 

Gene Expression Systems. One of the potentials of genetic engineering of microbes is production of large amounts of recombinant proteias (12,13). This is not a trivial task. Each proteia is unique and the stabiUty of the proteia varies depending on the host. Thus it is not feasible to have a single omnipotent microbial host for the production of all recombinant proteias. Rather, several microbial hosts have to be studied. Expression vectors have to be tailored to the microbe of choice. 

Keasling, J.D. (1999) Gene-expression tools for the metabolic engineering of bacteria. Trends in Biotechnology, 17, 452-460. 

Measurements of gene expression or protein abundance, which are often favored in metabolic engineering, do not necessarily provide a route to understand what is occurring within metabolic systems. 

Microbalances, 26 245 Microballoons, phenolic, 18 796, 797 Microbe genetic engineering, 72 470-484 future of, 12 482 gene technologies in, 72 470-475 host systems for gene expression, 72 475—480... 

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