Escherichia coli strains that allow antibiotic-free plasmid selection and maintenance by repressor titration

We report the construction of two novel Escherichia coli strains (DH1lacdapD and DH1lacP2dapD) that facilitate the antibiotic-free selection and stable maintenance of recombinant plasmids in complex media. They contain the essential chromosomal gene, dapD, under the control of the lac operator/promoter. Unless supplemented with IPTG (which induces expression of dapD) or DAP, these cells lyse. However, when the strains are transformed with a multicopy plasmid containing the lac operator, the operator competitively titrates the LacI repressor and allows expression of dapD from the lac promoter. Thus transformants can be isolated and propagated simply by their ability to grow on any medium by repressor titration selection. No antibiotic resistance genes or other protein expressing sequences are required on the plasmid, and antibiotics are not necessary for plasmid selection, making these strains a valuable tool for therapeutic DNA and recombinant protein production. We describe the construction of these strains and demonstrate plasmid selection and maintenance by repressor titration, using the new pORT plasmid vectors designed to facilitate recombinant DNA exploitation.

Titration of chaos with added noise

Deterministic chaos has been implicated in numerous natural and man-made complex phenomena ranging from quantum to astronomical scales and in disciplines as diverse as meteorology, physiology, ecology, and economics. However, the lack of a definitive test of chaos vs. random noise in experimental time series has led to considerable controversy in many fields. Here we propose a numerical titration procedure as a simple “litmus test” for highly sensitive, specific, and robust detection of chaos in short noisy data without the need for intensive surrogate data testing. We show that the controlled addition of white or colored noise to a signal with a preexisting noise floor results in a titration index that: (i) faithfully tracks the onset of deterministic chaos in all standard bifurcation routes to chaos; and (ii) gives a relative measure of chaos intensity. Such reliable detection and quantification of chaos under severe conditions of relatively low signal-to-noise ratio is of great interest, as it may open potential practical ways of identifying, forecasting, and controlling complex behaviors in a wide variety of physical, biomedical, and socioeconomic systems.

The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme.

Beta-Lactamases are widespread in the bacterial world, where they are responsible for resistance to penicillins, cephalosporins, and related compounds, currently the most widely used antibacterial agents. Detailed structural and mechanistic understanding of these enzymes can be expected to guide the design of new antibacterial compounds resistant to their action. A number of high-resolution structures are available for class A beta-lactamases, whose catalytic mechanism involves the acylation of a serine residue at the active site. The identity of the general base which participates in the activation of this serine residue during catalysis has been the subject of controversy, both a lysine residue and a glutamic acid residue having been proposed as candidates for this role. We have used the pH dependence of chemical modification of epsilon-amino groups by 2,4,6,-trinitrobenzenesulfonate and the pH dependence of the epsilon-methylene 1H and 13C chemical shifts (in enzyme selectively labeled with [epsilon-13C]lysine) to estimate the pKa of the relevant lysine residue, lysine-73, of TEM-1 beta-lactamase. Both methods show that the pKa of this residue is > 10, making it very unlikely that this residue could act as a proton acceptor in catalysis. An alternative mechanism in which this role is performed by glutamate-166 through an intervening water molecule is described.

Nonionic detergents in nonaqueous solvents,

The behavior of [alpha]-monoglycerides.

Uber die Volhardsche Methode der quantitativen Pepsin- und Trypsinbestimmung dur Titration /

Thesis (doctoral)--Grossherzogl. Hessische Landes- Universitat Giessen, 1905.

Noncovalent coatings for the separation of synthetic polypeptides by nonaqueous capillary electrophoresis

Recently, we demonstrated the possibility to extend the range of capillary electrophoresis (CE) applications to the separation of non-water-soluble synthetic polymers. This work focuses on the control of the electro-osmotic flow (EOF) and on the limitation of the solute adsorption in nonaqueous electrolytes. For these purposes, different strategies were investigated. For the initial, a viscous additive (ethylene glycol or glycerol) was used in the electrolyte in order to decrease the EOF magnitude and, possibly, to compete with solute adsorption. A second strategy was to modify, before separation, the fused-silica capillary wall by the adsorption of poly(ethylene oxide) (PEO) via hydrogen bonding. The influence of the molecular mass of the adsorbed PEO on the EOF magnitude and direction was studied in electrolytes based on methanol/acetonitrile mixtures containing ammonium ions. For PEO molecular masses above 1000 g/mol, reversed (anodic) EOF were reported in accordance with previous results obtained with PEO covalently bonded capillaries. The influence of the nature and the concentration of the background electrolyte cation on the EOF magnitude and direction were also investigated. A third strategy consisted in modifying the capillary wall by the adsorption of a cationic polyelectrolyte layer. Advantageously, this polyelectrolyte layer suppressed the adsorption of the polymer solutes onto the capillary wall. The results obtained in this work confirm the high potential and the versatility of CE for the characterization of ionizable organic polymers in nonaqueous media.

Nonaqueous capillary zone electrophoresis of synthetic organic polypeptides

Poly(Nε-trifluoroacetyl-l-lysine) was used as a model solute to investigate the potential of nonaqueous capillary electrophoresis (NACE) for the characterization of synthetic organic polymers. The information obtained by NACE was compared to that derived from size exclusion chromatography (SEC) experiments, and the two techniques were found to be complimentary for polymer characterization. On one hand, NACE permitted (i) the separation of oligomers according to their molar mass and (ii) the separation of the polymers according to the nature of the end groups. On the other hand, SEC experiments were used for the characterization of the molar mass distribution for higher molar masses. Due to the tendency of the solutes (polypeptides) to adsorb onto the fused-silica capillary wall, careful attention was paid to the rinsing procedure of the capillary between runs in order to keep the capillary surface clean. For that purpose, the use of electrophoretic desorption under denaturating conditions was very effective. Optimization of the separation was performed by studying (i) the influence of the proportion of methanol in a methanol/acetonitrile mixture and (ii) the influence of acetic acid concentration in the background electrolyte. Highly resolved separation of the oligomers (up to a degree of polymerization n of ∼50) was obtained by adding trifluoroacetic acid to the electrolyte. Important information concerning the polymer conformations could be obtained from the mobility data. Two different plots relating the effective mobility data to the degree of polymerization were proposed for monitoring the changes in polymer conformations as a function of the number of monomers.

Self-titration by experienced e-cigarette users: blood nicotine delivery and subjective effects

Rationale Self-titration is well documented in the tobacco literature. The extent to which ecigarette users (vapers) self-titrate is unknown. Objective This study explored the effects of high and low nicotine strength liquid on puffing topography, nicotine delivery and subjective effects in experienced vapers. Methods Eleven experienced male vapers completed 60 minutes of ad libitum vaping under low (6 mg/mL) and high (24 mg/mL) nicotine liquid conditions in two separate sessions. Measurements included: puffing topography (puff number, puff duration, volume of liquid consumed); and changes in: plasma nicotine levels, craving, withdrawal symptoms, selfreported hit, satisfaction and adverse effects. Results Liquid consumption and puff number were higher, and puff duration longer, in the low nicotine strength condition (all ps < 0.01). The mean difference in nicotine boost from baseline in the low condition was 8.59 (7.52) ng/mL, 16.99 (11.72) ng/mL and 22.03 (16.19) ng/mL at 10, 30 and 60 minutes respectively. Corresponding values for the high condition were 33.77 (34.88) ng/mL, 35.48 (28.31) ng/mL and 43.57 (34.78) ng/mL (ps < 0.05). There were no statistically significant differences between conditions in self-reported craving, withdrawal symptoms, satisfaction, hit or adverse effects. Conclusions Vapers engaged in compensatory puffing with lower nicotine strength liquid, doubling their consumption. Whilst compensatory puffing was sufficient to reduce craving and withdrawal discomfort, self-titration was incomplete with significantly higher plasma nicotine levels in the high condition.