Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay.

The recent discovery of riboswitches in diverse species of bacteria and few eukaryotes added metabolite-responsive gene regulation to the growing list of RNA functions in biology. The natural riboswitches have inspired several designs of synthetic analogues capable of gene regulation in response to a small molecule trigger. In this work, we describe our efforts to engineer complex riboswitches capable of sensing and responding to two small molecules according to Boolean logics AND and NAND. Two aptamers that recognize theophylline and thiamine pyrophosphate were embedded in tandem in the 5' UTR of bacterial mRNA, and riboswitches that function as logic gates were isolated by dual genetic selection. The diverse phenotype of the engineered logic gates supports the versatility of RNA-based gene regulation which may have preceded the modern protein-based gene regulators. Additionally, our design strategy advances our ability to harness the versatile capacities of RNA to program complex behavior in bacteria without the use of engineered proteins.

It has become increasingly evident that noncoding small RNAs (sRNAs) play a significant and global role in bacterial gene regulation. A majority of the trans-acting sRNAs in bacteria interact with the 5' untranslated region (UTR) and/or the translation initiation region of the targeted mRNAs via imperfect base pairing, resulting in reduced translation efficiency and/or mRNA stability. Additionally, bacterial sRNAs often contain distinct scaffolds that recruit RNA chaperones such as Hfq to facilitate gene regulation. In this study, we describe a strategy to engineer artificial sRNAs that can regulate desired endogenous genes in Escherichia coli. Using a fluorescent reporter gene that was translationally fused to a native 5' mRNA leader sequence, active artificial sRNAs were screened from libraries in which natural sRNA scaffolds were fused to a randomized antisense domain. Artificial sRNAs that posttranscriptionally repress two endogenous genes ompF and fliC were isolated and characterized. We anticipate that the artificial sRNAs will be useful for dynamic control and fine-tuning of endogenous gene expression in bacteria for applications in synthetic biology.

The presence of 4-terpineol, δ-3-carene, trans-sabinene hydrate, 1-phellandrene, α-terpinene, 1-terpineol, bicyclogermacrene, isopinocarveol, ledenoxid-II, longifolenaldehyde and α-terpenyl acetate make the aroma of the oil different from large cardamom oil of Sikkim and could offer potential as a new food flavour.

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Two transmembrane proteins were tentatively classified as NarK1 and NarK2 in the Pseudomonas genome project and hypothesized to play an important physiological role in nitrate/nitrite transport in Pseudomonas aeruginosa. The narK1 and narK2 genes are located in a cluster along with the structural genes for the nitrate reductase complex. Our studies indicate that the transcription of all these genes is initiated from a single promoter and that the gene complex narK1K2GHJI constitutes an operon. Utilizing an isogenic narK1 mutant, a narK2 mutant, and a narK1K2 double mutant, we explored their effect on growth under denitrifying conditions. While the ⌬narK1::Gm mutant was only slightly affected in its ability to grow under denitrification conditions, both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants were found to be severely restricted in nitratedependent, anaerobic growth. All three strains demonstrated wild-type levels of nitrate reductase activity. Nitrate uptake by whole-cell suspensions demonstrated both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants to have very low yet different nitrate uptake rates, while the ⌬narK1::Gm mutant exhibited wild-type levels of nitrate uptake. Finally, Escherichia coli narK rescued both the ⌬narK2::Gm and ⌬narK1K2::Gm mutants with respect to anaerobic respiratory growth. Our results indicate that only the NarK2 protein is required as a nitrate/nitrite transporter by Pseudomonas aeruginosa under denitrifying conditions. Denitrification involves four separate nitrogen oxide reductases and ultimately reduces nitrate to dinitrogen (37). Respiratory nitrate reductase, which is the first enzyme in this denitrification pathway, has its active site on the cytoplasmic side of the membrane (23). The enzyme substrate, nitrate, is an ion and cannot be taken up by the simple process of passive diffusion (18). Both of these factors require the bacterium to synthesize a transport protein(s) to carry nitrate into the cytoplasm, where the reduction of nitrate to nitrite takes place. It has been demonstrated for Pseudomonas aeruginosa, Pseudomonas stutzeri, and Escherichia coli (7,11,24) that the product of nitrate respiration, i.e., nitrite, is immediately excreted to the external environment, presumably protecting the organism from potential toxic effects. These toxic effects are due to the ability of this anion to bind to the heme groups in electron carriers, thereby inhibiting the flow of electrons (25). Genetic and physiological data suggest that nitrate transport in some bacteria occurs through two different uptake systems. Thus, for the process of nitrate assimilation, ABC transporters as well as secondary transporters are postulated to be used. On the other hand, anaerobically, for the purpose of nitrate respiration, it is postulated that bacteria rely solely on secondary transporters (18).Originally, John (14) demonstrated that membrane permeabilization of the cells significantly enhanced nitrate uptake, suggesting the need for a transport protein specific for nitrate. This was corroborated by sever...

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