A Forward-Design Approach to Increase the Production of Poly-3-Hydroxybutyrate in Genetically Engineered Escherichia coli

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB)

Multilevel Regulation and Translational Switches in Synthetic Biology

In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules.Thisisabouttochangeastherepertoireoffoundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and ribonucleic acid (RNA) responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications

Fabrication of SERS Active Surface on Polyimide Sample by Excimer Laser Irradiation

A possible application of excimer laser irradiation for the preparation of surface enhanced Raman spectroscopy (SERS) substrate is demonstrated. A polyimide foil of 125 μm thickness was irradiated by 240 pulses of focused ArF excimer laser beam (λ = 193 nm, FWHM = 20 ns). The applied fluence was varied between 40 and 80 mJ/cm2. After laser processing, the sample was coated with 40 nm silver by PLD in order to create a conducting layer required for the SERS application. The SERS activity of the samples was tested by Raman microscopy. The Raman spectra of Rhodamine 6G aqueous solution (c=10−3 mol/dm3) were collected from the patterned and metalized areas. For areas prepared at 40–60 mJ/cm2 laser fluences, the measured Raman intensities have shown a linear dependence on the applied laser fluence, while above 60 mJ/cm2 saturation was observed. The morphology of the SERS active surface areas was investigated by scanning electron microscopy. Finite element modeling was performed in order to simulate the laser-absorption induced heating of the polyimide foil. The simulation resulted in the temporal and spatial distribution of the estimated temperature in the irradiated polyimide sample, which are important for understanding the structure formation process

Crater formation by fast ions: comparison of experiment with Molecular Dynamics simulations

An incident fast ion in the electronic stopping regime produces a track of excitations which can lead to particle ejection and cratering. Molecular Dynamics simulations of the evolution of the deposited energy were used to study the resulting crater morphology as a function of the excitation density in a cylindrical track for large angle of incidence with respect to the surface normal. Surprisingly, the overall behavior is shown to be similar to that seen in the experimental data for crater formation in polymers. However, the simulations give greater insight into the cratering process. The threshold for crater formation occurs when the excitation density approaches the cohesive energy density, and a crater rim is formed at about six times that energy density. The crater length scales roughly as the square root of the electronic stopping power, and the crater width and depth seem to saturate for the largest energy densities considered here. The number of ejected particles, the sputtering yield, is shown to be much smaller than simple estimates based on crater size unless the full crater morphology is considered. Therefore, crater size can not easily be used to estimate the sputtering yield.Comment: LaTeX, 7 pages, 5 EPS figures. For related figures/movies, see: http://dirac.ms.virginia.edu/~emb3t/craters/craters.html New version uploaded 5/16/01, with minor text changes + new figure

Comparison of three nanoparticle deposition techniques potentially applicable to elemental mapping by nanoparticle-enhanced laser-induced breakdown spectroscopy

In this study, we compared the applicability of three nanoparticle deposition techniques (spray coating, spark discharge nanoparticle generation, magnetron sputtering) towards elemental mapping by nanoparticle-enhanced laser-induced breakdown spectroscopy (NE-LIBS). It was found that sputtering followed by a thermal treatment at 550 °C can provide a homogenous, practical and controllable way of NE-LIBS sample preparation with gold nanoparticles. The laser ablation properties of the created NP layer was also studied in detail and it was established that a 200 μm laser spot size is good compromise between the NE-LIBS signal enhancement and the spatial resolution required for mapping. A signal enhancement of about a factor of 10 with good repeatability (ca. 5 %RSD) in a line scanning demonstration was achieved on glass for Si detection. For samples that are fairly temperature and vacuum stable, this approach allows the signal enhancement to be used in mapping applications

Qualitative Analysis of Glass Microfragments Using the Combination of Laser-Induced Breakdown Spectroscopy and Refractive Index Data

We have successfully demonstrated that although there are significant analytical challenges involved in the qualitative discrimination analysis of sub-mm sized (microfragment) glass samples, the task can be solved with very good accuracy and reliability with the multivariate chemometric evaluation of laser-induced breakdown spectroscopy (LIBS) data or in combination with pre-screening based on refractive index (RI) data. In total, 127 glass samples of four types (fused silica, flint, borosilicate and soda–lime) were involved in the tests. Four multivariate chemometric data evaluation methods (linear discrimination analysis, quadratic discrimination analysis, classification tree and random forest) for LIBS data were evaluated with and without data compression (principal component analysis). Classification tree and random forest methods were found to give the most consistent and most accurate results, with classifications/identifications correct in 92 to 99% of the cases for soda–lime glasses. The developed methods can be used in forensic analysis

Desorption of nanoclusters (2\u201340 nm) from nanodispersed metal and semiconductor layers by swift heavy ions

New experimental data on the desorption of nanoclusters (2\u201340 nm) from nanodispersed layers of metals (Ag, Pt, In, Bi) and semiconductors (Ge, UO2, PbS) by fission fragments (FF) are reported and compared to the previously obtained data on desorption of nanoclusters of gold. It is shown that the desorption of nanoclusters from nanodispersed layers of metals and semiconductors due to electronic processes induced by swift heavy ions is a universal phenomenon. The yield of the desorbed nanoclusters depends on the cluster material and cluster size as well as on the material of the target substrate and varies within the range from 3c5 to 3c0.001 FF 121. The angular distributions of the nanoclusters do not depend on the cluster material, but only on the cluster size: the mean polar angle of desorption decreasing from 45\ub0 to 23\ub0 while cluster size increasing from 2 to 40 nm. The majority of the nanoclusters is desorbed in the form of molten, liquid droplets. The results are discussed