Stepwise optimization of recombinant protein production in Escherichia coli utilizing computational and experimental approaches.

Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.

Electrospun Silver Coated Polyacrylonitrile Membranes for Water Filtration Applications.

The scarcity of drinking water and the contamination of water sources in underdeveloped countries are serious problems that require immediate low-tech and low-cost solutions. In this study, we fabricated polyacrylonitrile (PAN) porous membranes coated with silver nanoparticles (AgNP) and demonstrated their use for water filtration and water treatment applications. The membranes were prepared by electrospinning a PAN solution and treating in a hydroxylamine (NH2OH) aqueous solution to form ⁻C(NH2)N⁻OH groups that were used for functionalization (Ag⁺ ions) of the membrane. The coordinated silver ions were then converted to silver nanoparticles. The microstructure of the membrane, water permeability, antimicrobial effect (using Escherichia coli), and particulate filtration capabilities were studied. This study verified that the membrane demonstrated a 100% reduction for Gram-negative bacteria with an effective filtration rate of 8.0 mL/cm² min. Furthermore, the membrane was able to eliminate 60% of latex beads as small as 50 nm and over 80% of the 2 µm beads via gravity filtration. This study demonstrated that PAN⁻AgNP membranes can be employed as antimicrobial membranes for the filtration of water in underdeveloped countries.

Survey of natural and transgenic gene markers used to monitor transposon activity.

Marker genes have played a critical role in the discovery of plant transposable elements, our understanding of transposon biology, and the utility of transposable elements as tools in functional genomics. Marker traits in model plants have been useful to detect transposable elements and to study the dynamics of transposition. Transposon-induced changes in the sequence of marker genes and consequently their expression have contributed to our understanding of molecular mechanisms of transposition and associated genome rearrangements. Further, marker genes that have been cloned and are compatible in heterologous systems have found versatile utility in the design of DNA constructs that have enabled us to understand the finer details of transposition mechanisms, and also allowed the use of transposon-based tools for functional genomics. This chapter traces the role of marker traits and marker genes (endogenous and transgenic) in various plant systems, and their contributions to the advancement of transposon biology over the past several decades.

In vivo modification of a maize engineered minichromosome.

Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

Synthetic chromosome platforms in plants.

Synthetic chromosomes provide the means to stack transgenes independently of the remainder of the genome. Combining them with haploid breeding could provide the means to transfer many transgenes more easily among varieties of the same species. The epigenetic nature of centromere formation complicates the production of synthetic chromosomes. However, telomere-mediated truncation coupled with the introduction of site-specific recombination cassettes has been used to produce minichromosomes consisting of little more than a centromere. Methods that have been developed to modify genes in vivo could be applied to minichromosomes to improve their utility and to continue to increase their length and genic content. Synthetic chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agricultural applications or to use plants as factories for the production of foreign proteins or metabolites.

Engineered plant minichromosomes.

The advent of transgenic technologies has met many challenges, both technical and political; however, these technologies are now widely applied, particularly for crop improvement. Bioengineering has resulted in plants carrying resistance to herbicides, insects, and viruses, as well as entire biosynthetic pathways. Some of the technical challenges in generating transgenic plant or animal materials include: an inability to control the location and nature of the integration of transgenic DNA into the host genome, and linkage of transformed genes to selectable antibiotic resistance genes used in the production of the transgene cassette. Furthermore, successive transformation of multiple genes may require the use of several selection genes. The coordinated expression of multiple stacked genes would be required for complex biosynthetic pathways or combined traits. Engineered nonintegrating minichromosomes can overcome many of these problems and hold much promise as key players in the next generation of transgenic technologies for improved crop plants. In this review, we discuss the history of artificial chromosome technology with an emphasis on engineered plant minichromosomes.

Transposable elements as catalysts for chromosome rearrangements.

Barbara McClintock first showed that transposable elements in maize can induce major chromosomal rearrangements, including duplications, deletions, inversions, and translocations. More recently, researchers have made significant progress in elucidating the mechanisms by which transposons can induce genome rearrangements. For the Ac/Ds transposable element system, rearrangements are generated when the termini of different elements are used as substrates for transposition. The resulting alternative transposition reaction directly generates a variety of rearrangements. The size and type of rearrangements produced depend on the location and orientation of transposon insertion. A single locus containing a pair of alternative transposition-competent elements can produce a virtually unlimited number of genome rearrangements. With a basic understanding of the mechanisms involved, researchers are beginning to utilize both naturally occurring and in vitro-generated configurations of transposable elements in order to manipulate chromosome structure.

Fusion of reverse-oriented Ds termini following abortive transposition in Arabidopsis: implications for the mechanism of Ac/Ds transposition.

We studied the products of alternative transposition reactions that utilize reverse-oriented Ds termini as substrates. In this configuration, Ds transposition can generate genome rearrangements including deletions, inversions, and reciprocal translocations. In approximately half of the transposition products recovered in Arabidopsis, the termini of the reversed ends Ds element were ligated together. The sequences at these fused-end junctions suggest that the excised transposon termini form covalently closed hairpin structures. These results shed new light on the mechanism of Ac/Ds transposition.

Reversed end Ds element: a novel tool for chromosome engineering in Arabidopsis.

The maize Ac/Ds transposable element (TE) transposes by a "cut and paste" mechanism. Previous studies in maize showed that when the TE ends are in reversed orientation with respect to each other, alternative transposition reactions can occur resulting in large scale genome rearrangements including deletions and inversions. To test whether similar genome rearrangements can also occur in other plants, we studied the efficacy of such alternative transposition-mediated genome rearrangements in Arabidopsis. Here we present our analysis of 33 independent chromosome rearrangements. Transposition at the reversed ends Ds element can cause deletions over 1 Mbp, and inversions up to 2.4 Mbp in size. We identified additional rearrangements including a reciprocal translocation and a putative ring chromosome. Some of the deletions and inversions are germinally transmitted.

Fold prediction and comparative modeling of Bdm1: a probable alpha/beta hydrolase associated with hot water epilepsy.

Hot water epilepsy (HWE) is a benign and rare form of reflex epilepsy that occurs most commonly in humans. Bdm1 is one of the proteins whose mRNA transcript is overexpressed during HWE in a rat model. We show, by sequence analysis and fold recognition methods, that Bdm1 has strong structural similarities to alpha/beta hydrolases like the thioesterases. A three-dimensional model derived by comparative modeling methods allowed the search for catalytic residues using a flexible functional template characteristic of these enzymes. We predict that Bdm1 might be regulated by homocysteine levels by means of direct participation in degradation pathways.