Plasmid DNA (pDNA) is an emerging experimental vaccine, produced in E. coli, initially targeted for viral diseases. Unlike traditional protein vaccines whose average dose is micrograms, the average dose of pDNA is on the scale of milligrams. Production yields are, therefore, important for the future development of this vaccine. The E. coli strains currently used for pDNA production, JM109 and DH5alpha, are both suitable for production of stable pDNA due to the deletion of recA and endA, however, these two E. coli K strains are sensitive to growth conditions such as high glucose concentration. On the other hand E. coli BL21 is less sensitive to growth conditions than E. coli JM109 or DH5alpha, this strain grows to higher densities and due to its active glyoxylate shunt and anaplerotic pathways is not sensitive to high glucose concentration. This strain is used for recombinant protein production but not for pDNA production because of its inability to produce stable pDNA. To adapt E. coli BL21 for stable pDNA production, the strain was mutated by deleting both recA and endA, and a proper growth and production strategy was developed. Production values, reaching 2 g/L were obtained using glucose as a carbon source. The produced plasmid, which was constructed for HIV clinical study, was found to have identical properties to the plasmid currently produced by E. coli DH5alpha.

Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated endproducts, respectively. Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We report that a perturbation in the critical step of a metabolic pathway-the D-galactose amphibolic pathwaychanges the dynamics of the pathways leading to accumulation of the intermediary metabolite UDP-galactose. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis). Such studies are an essential component in the integration of metabolomics, proteomics, and transcriptomics.galactose metabolism ͉ gene signals ͉ intracellular stress ͉ tiling arrays A cell is capable of carrying out thousands of chemical reactions to make or break compounds of different kinds (metabolites). To achieve proper balance in its chemical constituents, these reactions are programmed in time. If there is either a lack or an excess of an important metabolite, it could create stress. The cell is expected to take care of such a stress in two ways: (i) The first is a quick fix, which occurs at a biochemical level by activation or inhibition of the catalytic activities of enzymes by metabolites so as to restore homeostasis to the chemical milieu of the cell. (ii) The second is a long term solution in which the synthesis of enzymes or proteins whose presence or absence causes the chemical imbalance is turned off or on. These control mechanisms occur at different levels, but the primary level of a metabolite signal is perceived at the level of transcription. So far, the literature describes two classes of metabolites that signal gene transcription: (i) a substrate of an enzymatic pathway induces the synthesis of the enzymes of that pathway (usually catabolic), or (ii) the end-product of a pathway represses the synthesis of the enzymes of the pathway (usually anabolic). However, except for protein-modifying metabolites, e.g., acetylCoA (1, 2) or acetyl-phosphate (3), intermediary metabolites signaling even a specific gene transcription have been reported only in very few cases (4-6). We propose that gene regulation mediated by intermediary metabolites may be more widespread than previously perceived, particularly with respect to the regulation of genes encoding enzymes of amphibolic pathways, in which catabolic pathways overlap with or connect to anabolic pathways. Knowledge of the nature and extent of this type of regulation in an organism is needed for a complete understanding of the genetic regulatory network of cells. We explored one such metabolic intermediate, UDP-galactose, which we found not only sends signals to specific genes to...

Using recombinant DNA technology, various whole-cell biosensors have been developed for detection of environmental pollutants, including heavy metal ions. Whole-cell biosensors have several advantages: easy and inexpensive cultivation, multiple assays, and no requirement of any special techniques for analysis. In the era of synthetic biology, cutting-edge DNA sequencing and gene synthesis technologies have accelerated the development of cell-based biosensors. Here, we summarize current technological advances in whole-cell heavy metal biosensors, including the synthetic biological components (bioparts), sensing and reporter modules, genetic circuits, and chassis cells. We discuss several opportunities for improvement of synthetic cell-based biosensors. First, new functional modules must be discovered in genome databases, and this knowledge must be used to upgrade specific bioparts through molecular engineering. Second, modules must be assembled into functional biosystems in chassis cells. Third, heterogeneity of individual cells in the microbial population must be eliminated. In the perspectives, the development of whole-cell biosensors is also discussed in the aspects of cultivation methods and synthetic cells.

Individual deletions of acs and aceA genes in E. coli B (BL21) showed little difference in the metabolite accumulation patterns but deletion of the ackA gene alone or together with pta showed acetic acid gradually accumulated to 3.1 and 1.7 g/l, respectively, with a minimal extended lag in bacterial growth and a higher pyruvate formation. Single poxB deletion in E. coli B (BL21) or additional poxB deletion in the ackA-pta mutants did not change the acetate accumulation pattern. When the acetate production genes (ackA-pta-poxB) were deleted in E. coli B (BL21) acetate still accumulated. This may be an indication that perhaps acetate is not only a by-product of carbon metabolism; it is possible that acetate plays also a role in other cellular metabolite pathways. It is likely that there are alternative acetate production pathways.