Biological validation of a novel process and product for quantitating western blots.

Protein normalization of western blots has relied upon housekeeping proteins which exhibit signal saturation and varied cellular expression level variations. These issues can produce spurious results leading to erroneous conclusions. A superior method to protein normalization using housekeeping proteins is Total Protein Normalization, a method now recognized as the gold standard for quantitative westerns. Total Protein Normalization requires that all proteins on a membrane be stained or labeled uniformly, imaged, and then analyzed for total protein. It is important that such a normalization process not interfere with typical immunodetection methods, fits within existing western workflows, and exhibits a linear relationship of signal intensity to protein load under all experimental conditions. Here we report that we developed a new reagent enabling Total Protein Normalization, and we demonstrate its superior protein normalization capabilities through analysis of target proteins in different cell backgrounds. These data illustrate how housekeeping proteins exhibit signal saturation, yield erroneous normalization data, and display sample-to-sample variations averaging 48.2 % overall. Signal intensities obtained using our new method show a linear relationship to protein sample load, thus providing accurate protein normalization with an overall average variation of 7.7 %.

An integrated biotechnology platform for developing sustainable chemical processes.

Genomatica has established an integrated computational/experimental metabolic engineering platform to design, create, and optimize novel high performance organisms and bioprocesses. Here we present our platform and its use to develop E. coli strains for production of the industrial chemical 1,4-butanediol (BDO) from sugars. A series of examples are given to demonstrate how a rational approach to strain engineering, including carefully designed diagnostic experiments, provided critical insights about pathway bottlenecks, byproducts, expression balancing, and commercial robustness, leading to a superior BDO production strain and process.

Evaluation of exposure to airborne heavy metals at gun shooting ranges.

Aerosols formed during shooting events were studied with various techniques including the wide range size resolving sampling system Nano-ID(®) Select, followed by inductively coupled plasma mass spectrometry chemical analysis, scanning electron microscopy, and fast mobility particle sizing. The total lead mass aerosol concentration ranged from 2.2 to 72 µg m(-3). It was shown that the mass concentration of the most toxic compound lead is much lower than the total mass concentration. The deposition fraction in various compartments of the respiratory system was calculated using the ICRP lung deposition model. It was found that the deposition fraction in the alveolar range varies by a factor >3 for the various aerosols collected, depending on the aerosol size distribution and total aerosol concentration, demonstrating the importance of size resolved sampling in health risk evaluation. The proportion of the total mass of airborne particles deposited in the respiratory tract varies from 34 to 70%, with a median of 55.9%, suggesting the health risk based upon total mass significantly overestimates the accumulated dose and therefore the health risk. A comparison between conventional and so called 'green' ammunition confirmed significant lowering of concentrations of lead and other toxic metals like antimony in the atmosphere of indoor shooting ranges using 'green' ammunition, although higher concentrations of manganese and boron were measured. These metals are likely to be the constituents of new types of primers. They occur predominantly in the size fraction <250 nm of aerosols.

Surface area controlled heterogeneous nucleation.

Heterogeneous nucleation of liquid from a gas phase on nanoparticles has been studied under various saturation ratios and nuclei size. The probability of liquid droplet nucleation, especially at a low degree of deviation from equilibrium, was measured for both atmospheric aerosol particles and engineered nanoparticles Cr(2)O(3). The concept of a critical saturation ratio and the validity of the one-to-one relationship between the nuclei number and the number of droplets were examined. A transient zone between no nucleation and established nucleation termed the surface area controlled nucleation was observed. In this zone, the probability of stable phase formation is determined by the surface area of nuclei. There are two distinctive features of the surface area controlled nucleation: the nucleation probability is much less than 1 and is proportional to the surface area of nuclei. For condensation particle counters (CPCs) counting nanoparticles, these features mean that counts measured are proportional to the surface area of nanoparticles and, therefore, the CPCs counts can be calibrated to measure the surface area.

A multifunctional hybrid glycosyl hydrolase discovered in an uncultured microbial consortium from ruminant gut.

A unique multifunctional glycosyl hydrolase was discovered by screening an environmental DNA library prepared from a microbial consortium collected from cow rumen. The protein consists of two adjacent catalytic domains. Sequence analysis predicted that one domain conforms to glycosyl hydrolase family 5 and the other to family 26. The enzyme is active on several different beta-linked substrates and possesses mannanase, xylanase, and glucanase activities. Site-directed mutagenesis studies on the catalytic residues confirmed the presence of two functionally independent catalytic domains. Using site-specific mutations, it was shown that one catalytic site hydrolyzes beta-1,4-linked mannan substrates, while the second catalytic site hydrolyzes beta-1,4-linked xylan and beta-1,4-linked glucan substrates. Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE) also confirmed that the enzyme has discrete domains for binding and hydrolysis of glucan- and mannan-linked polysaccharides. Such multifunctional enzymes have many potential industrial applications in plant processing, including biomass saccharification, animal feed nutritional enhancement, textile, and pulp and paper processing.

Unusual microbial xylanases from insect guts.

Recombinant DNA technologies enable the direct isolation and expression of novel genes from biotopes containing complex consortia of uncultured microorganisms. In this study, genomic libraries were constructed from microbial DNA isolated from insect intestinal tracts from the orders Isoptera (termites) and Lepidoptera (moths). Using a targeted functional assay, these environmental DNA libraries were screened for genes that encode proteins with xylanase activity. Several novel xylanase enzymes with unusual primary sequences and novel domains of unknown function were discovered. Phylogenetic analysis demonstrated remarkable distance between the sequences of these enzymes and other known xylanases. Biochemical analysis confirmed that these enzymes are true xylanases, which catalyze the hydrolysis of a variety of substituted beta-1,4-linked xylose oligomeric and polymeric substrates and produce unique hydrolysis products. From detailed polyacrylamide carbohydrate electrophoresis analysis of substrate cleavage patterns, the xylan polymer binding sites of these enzymes are proposed.

Recent progress in biocatalyst discovery and optimization.

The use of enzymes in industrial catalysis continues to grow because of the considerable advantages of natural catalytic systems. The need for enantiomerically pure fine chemicals and the movement away from chemically burdened technologies will drive the acceptance of enzyme-assisted processes. New technologies for enzyme discovery and optimization have enabled the application of enzymes in harsh industrial conditions and in processes demanding stringent selectivity. These discovery and laboratory evolution methods entail genomic approaches that by their nature engender screening of extremely large numbers of gene types and variants. By extension, the fitness of an individual high-throughput screen requires an intelligent, process-targeted assay amenable to a chosen screening platform.

An evolutionary route to xylanase process fitness.

Directed evolution technologies were used to selectively improve the stability of an enzyme without compromising its catalytic activity. In particular, this article describes the tandem use of two evolution strategies to evolve a xylanase, rendering it tolerant to temperatures in excess of 90 degrees C. A library of all possible 19 amino acid substitutions at each residue position was generated and screened for activity after a temperature challenge. Nine single amino acid residue changes were identified that enhanced thermostability. All 512 possible combinatorial variants of the nine mutations were then generated and screened for improved thermal tolerance under stringent conditions. The screen yielded eleven variants with substantially improved thermal tolerance. Denaturation temperature transition midpoints were increased from 61 degrees C to as high as 96 degrees C. The use of two evolution strategies in combination enabled the rapid discovery of the enzyme variant with the highest degree of fitness (greater thermal tolerance and activity relative to the wild-type parent).