Results of the Mitrofanoff procedure in urinary tract reconstruction in children

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90139/1/j.1464-410X.1997.33724.x.pd

Design and application of genetically-encoded malonyl-CoA biosensors for metabolic engineering of microbial cell factories

Malonyl-CoA is the basic building block for synthesizing a range of important compounds including fatty acids, phenylpropanoids, flavonoids and non-ribosomal polyketides. Centering around malonyl-CoA, we summarized here the various metabolic engineering strategies employed recently to regulate and control malonyl-CoA metabolism and improve cellular productivity. Effective metabolic engineering of microorganisms requires the introduction of heterologous pathways and dynamically rerouting metabolic flux towards products of interest. Transcriptional factor-based biosensors translate an internal cellular signal to a transcriptional output and drive the expression of the designed genetic/biomolecular circuits to compensate the activity loss of the engineered biosystem. Recent development of genetically-encoded malonyl-CoA sensor has stood out as a classical example to dynamically reprogram cell metabolism for various biotechnological applications. Here, we reviewed the design principles of constructing a transcriptional factor-based malonyl-CoA sensor with superior detection limit, high sensitivity and broad dynamic range. We discussed various synthetic biology strategies to remove pathway bottleneck and how genetically-encoded metabolite sensor could be deployed to improve pathway efficiency. Particularly, we emphasized that integration of malonyl-CoA sensing capability with biocatalytic function would be critical to engineer efficient microbial cell factory. Biosensors have also advanced beyond its classical function of a sensor actuator for in situ monitoring of intracellular metabolite concentration. Applications of malonyl-CoA biosensors as a sensor-invertor for negative feedback regulation of metabolic flux, a metabolic switch for oscillatory balancing of malonyl-CoA sink pathway and source pathway and a screening tool for engineering more efficient biocatalyst are also presented in this review. We envision the genetically-encoded malonyl-CoA sensor will be an indispensable tool to optimize cell metabolism and cost-competitively manufacture malonyl-CoA-derived compounds

An engineered constitutive promoter set with broad activity range for Cupriavidus necator H16

Well-characterized promoters with variable strength form the foundation of heterologous pathway optimization. It is also a key element that bolsters the success of microbial engineering and facilitates the development of biological tools like biosensors. In comparison to microbial hosts such as Escherichia coli and Saccharomyces cerevisiae, the promoter repertoire of Cupriavidus necator H16 is highly limited. This limited number of characterized promoters poses a significant challenge during the engineering of C. necator H16 for biomanufacturing and biotechnological applications. In this article, we first examined the architecture and genetic elements of the four most widely used constitutive promoters of C. necator H16 (i.e., PphaC1, PrrsC, Pj5, and Pg25) and established a narrow 6-fold difference in their promoter activities. Next, using these four promoters as starting points and applying a range of genetic modifications (including point mutation, length alteration, incorporation of regulatory genetic element, promoter hybridization, and configuration alteration), we created a library of 42 constitutive promoters, all of which are functional in C. necator H16. Although these promoters are also functional in E. coli, they show different promoter strength and hierarchical rank of promoter activity. Subsequently, the activity of each promoter was individually characterized, using l-arabinose-inducible PBAD promoter as a benchmark. This study has extended the range of constitutive promoter activities to 137-fold, with some promoter variants exceeding the l-arabinose-inducible range of PBAD promoter. Not only has the work enhanced our flexibility in engineering C. necator H16, it presented novel strategies in adjusting promoter activity in C. necator H16 and highlighted similarities and differences in transcriptional activity between this organism and E. coli

An efficient transformation method for the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16

Ralstonia eutropha H16 (also known as Cupriavidus necator H16) is a Gram-negative lithoautotrophic β-proteobacterium with increasing biotechnological applications, including carbon capture and utilization, biopolymer synthesis and biofuel production. Engineering of this organism is supported by the availability of its genome sequence and suitable plasmid systems. However, the lack of a simple and robust transformation method remains a challenge as it limits both the pace and ease of engineering this organism. To overcome this limitation, a systematic study was performed to evaluate the effects of different parameters on the transformation efficiency of R. eutropha H16. The optimized electroporation protocol uses R. eutropha H16 cells grown to OD600 0.6. These cells were made competent by a 15-min incubation in 50 mM CaCl2 , followed by two cell washes and final resuspension in 0.2 M sucrose prior to electroporation using 2.3 kV. This protocol achieved a transformation efficiency of (3.86 ± 0.29) x 10(5) cfu/μg DNA, a 10(3) -fold improvement compared to a previously published value for the same plasmid. This transformation method is a valuable tool for R. eutropha H16 research and will further enable the development of other advanced molecular biology methods for this industrially relevant microorganism

Accelerated directed evolution of dye-decolorizing peroxidase using a bacterial extracellular protein secretion system (BENNY)

Background Dye-decolorizing peroxidases (DyPs) are haem-containing peroxidases that show great promises in industrial biocatalysis and lignocellulosic degradation. Through the use of Escherichia coli osmotically-inducible protein Y (OsmY) as a bacterial extracellular protein secretion system (BENNY), we successfully developed a streamlined directed evolution workflow to accelerate the protein engineering of DyP4 from Pleurotus ostreatus strain PC15. Result After 3 rounds of random mutagenesis with error-prone polymerase chain reaction (epPCR) and 1 round of saturation mutagenesis, we obtained 4D4 variant (I56V, K109R, N227S and N312S) that displays multiple desirable phenotypes, including higher protein yield and secretion, higher specific activity (2.7-fold improvement in kcat/Km) and higher H2O2 tolerance (sevenfold improvement based on IC50). Conclusion To our best knowledge, this is the first report of applying OsmY to simplify the directed evolution workflow and to direct the extracellular secretion of a haem protein such as DyP4

Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli

Signal peptides and secretory carrier proteins are commonly used to secrete heterologous recombinant protein in Gram-negative bacteria. The Escherichia coli osmotically-inducible protein Y (OsmY) is a carrier protein that secretes a target protein extracellularly, and we have previously applied it in the Bacterial Extracellular Protein Secretion System (BENNY) to accelerate directed evolution. In this study, we reported the first application of random and combinatorial mutagenesis on a carrier protein to enhance total secretory target protein production. After one round of random mutagenesis followed by combining the mutations found, OsmY(M3) (L6P, V43A, S154R, V191E) was identified as the best carrier protein. OsmY(M3) produced 3.1 ± 0.3 fold and 2.9 ± 0.8 fold more secretory Tfu0937 β-glucosidase than its wildtype counterpart in E. coli strains BL21(DE3) and C41(DE3), respectively. OsmY(M3) also produced more secretory Tfu0937 at different cultivation temperatures (37 °C, 30 °C and 25 °C) compared to the wildtype. Subcellular fractionation of the expressed protein confirmed the essential role of OsmY in protein secretion. Up to 80.8 ± 12.2% of total soluble protein was secreted after 15 h of cultivation. When fused to a red fluorescent protein or a lipase from Bacillus subtillis, OsmY(M3) also produced more secretory protein compared to the wildtype. In this study, OsmY(M3) variant improved the extracellular production of three proteins originating from diverse organisms and with diverse properties, clearly demonstrating its wide-ranging applications. The use of random and combinatorial mutagenesis on the carrier protein demonstrated in this work can also be further extended to evolve other signal peptides or carrier proteins for secretory protein production in E. coli

Lepton Flavor Non-Conservation

In the present work we review the most prominent lepton flavor violating processes (\mu \ra e\gamma, \mu \ra 3e, $(\mu , e)$ conversion, $M-\bar M$ oscillations etc), in the context of unified gauge theories. Many currently fashionable extensions of the standard model are considered, such as: {\it i)} extensions of the fermion sector (right-handed neutrino); {\it ii)} minimal extensions involving additional Higgs scalars (more than one isodoublets, singly and doubly charged isosinglets, isotriplets with doubly charged members etc.); {\it iii)} supersymmetric or superstring inspired unified models emphasizing the implications of the renormalization group equations in the leptonic sector. Special attention is given to the experimentaly most interesting $(\mu - e)$ conversion in the presence of nuclei. The relevant nuclear aspects of the amplitudes are discussed in a number of fashionable nuclear models. The main features of the relevant experiments are also discussed, and detailed predictions of the above models are compared to the present experimental limits.Comment: (IOA-300/93, review article, 83p, 6 epsf figures , available upon request from [email protected])

Screening of cosmological constant for De Sitter Universe in non-local gravity, phantom-divide crossing and finite-time future singularities

We investigate de Sitter solutions in non-local gravity as well as in non-local gravity with Lagrange constraint multiplier. We examine a condition to avoid a ghost and discuss a screening scenario for a cosmological constant in de Sitter solutions. Furthermore, we explicitly demonstrate that three types of the finite-time future singularities can occur in non-local gravity and explore their properties. In addition, we evaluate the effective equation of state for the universe and show that the late-time accelerating universe may be effectively the quintessence, cosmological constant or phantom-like phases. In particular, it is found that there is a case in which a crossing of the phantom divide from the non-phantom (quintessence) phase to the phantom one can be realized when a finite-time future singularity occurs. Moreover, it is demonstrated that the addition of an $R^2$ term can cure the finite-time future singularities in non-local gravity. It is also suggested that in the framework of non-local gravity, adding an $R^2$ term leads to possible unification of the early-time inflation with the late-time cosmic acceleration.Comment: 42 pages, no figure, version accepted for publication in General Relativity and Gravitatio

Lepton Flavour Violating Leptonic/Semileptonic Decays of Charged Leptons in the Minimal Supersymmetric Standard Model

We consider the leptonic and semileptonic (SL) lepton flavour violating (LFV) decays of the charged leptons in the minimal supersymmetric standard model (MSSM). The formalism for evaluation of branching fractions for the SL LFV charged-lepton decays with one or two pseudoscalar mesons, or one vector meson in the final state, is given. Previous amplitudes for the SL LFV charged-lepton decays in MSSM are improved, for instance the $\gamma$-penguin amplitude is corrected to assure the gauge invariance. The decays are studied not only in the model-independent formulation of the theory in the frame of MSSM, but also within the frame of the minimal supersymmetric SO(10) model within which the parameters of the MSSM are determined. The latter model gives predictions for the neutrino-Dirac Yukawa coupling matrix, once free parameters in the model are appropriately fixed to accommodate the recent neutrino oscillation data. Using this unambiguous neutrino-Dirac Yukawa couplings, we calculate the LFV leptonic and SL decay processes assuming the minimal supergravity scenario. A very detailed numerical analysis is done to constrain the MSSM parameters. Numerical results for SL LFV processes are given, for instance for tau -> e (mu) pi0, tau -> e (mu) eta, tau -> e (mu) eta', tau -> e (mu) rho0, tau -> e (mu) phi, tau -> e (mu) omega, etc.Comment: 36 pages, 3 tables, 5 .eps figure