No abstract

The 3.31-Mb genome sequence of the intracellular pathogen and potential bioterrorism agent, Brucella suis, was determined. Comparison of B. suis with Brucella melitensis has defined a finite set of differences that could be responsible for the differences in virulence and host preference between these organisms, and indicates that phage have played a significant role in their divergence. Analysis of the B. suis genome reveals transport and metabolic capabilities akin to soil͞plant-associated bacteria. Extensive gene synteny between B. suis chromosome 1 and the genome of the plant symbiont Mesorhizobium loti emphasizes the similarity between this animal pathogen and plant pathogens and symbionts. A limited repertoire of genes homologous to known bacterial virulence factors were identified.

robotic workcell to conduct plasmid-based functional proteomics is being developed for optimization of protein open reading frames (ORF). The initial phase of this project is to design and assemble a Xantus liquid handler from Sias, Inc. modified by Hudson so that a workcell track component can be placed within the Xantus R gripper tool work area. The liquid handler is designed to produce plasmids using the Qiagen Turbo R plasmid preparation kit. This design allows processing of up to four 96-well plates in one run. The procedure eliminates disposable tips and provides an advanced wash system to prevent cross contamination. To evaluate liquid handler operation, a mutagenized cellulase F ORF plasmid library was prepared from wild-type cellulase F (Chen, H.; Li, X.-L.; Blum, D. L.; Ximenes, E. A.; Ljungdahl, L. G. CelF of Orpinomyces PC-2 has an intron and encodes a cellulase (CelF) containing a carbohydrate-binding module. Applied Biochemistry and Biotechnology 2003, 105-108, 775-785; Li, X.-L.; Chen, H.; Ljungdahl, L. G. Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase-hemicellulase complex. Applied and Environmental Microbiology 1997, 63(12), 4721-4728) using a novel Invitrogen Gateway R cloning strategy. For the automated reproducibility run, the average yield of plasmid was 5.35 mg per well from 1.347 mL of starting culture. Four plates were processed automatically on the liquid handler in 374 min compared to at least 441 min for the same plate operations performed manually. The quality and quantity of plasmids prepared on the liquid handler made the implementation of the following workcell protocols possible: DNA sequencing, in vitro transcription/translation, and transformation of bacterial and yeast strains for protein expression. ( JALA 2005;10:287-300)

Robotic platforms are essential for production of large numbers of expression-ready plasmid sets to develop optimized clones and improved microbial strains for crucial bioenergy applications and simultaneous high-value peptide expression. Here we demonstrate a plasmid-based integrated robotic workcell, modified with a motorized vacuum filtration system, for performing fully automated molecular biology protocols, including assembly of mutagenized gene sequences, purification of PCR amplicons, ligation of PCR products into vectors, transformation of competent Escherichia coli, plating of recovered transformants, plasmid preparation, cloning, and expression of optimized genes. A library of genes encoding variants of wolf spider lycotoxin-1, a candidate bioinsecticide, was produced using PCR mutagenesis in an amino acid scanning strategy to generate a complete set of mutations across the lycotoxin-1 gene. The improved vacuum filtration system permits automated purification of PCR products. Methods for recovery and growth of bacteria containing plasmids with PCR inserts allow individual colony formation on a novel solid medium in a deepwell plate. Inserts are cloned into a bacterial vector to verify expression. These protocols form the core of a fully automated molecular biology platform that reduces the cost and time required to perform all operations. (JALA 2007;12:202–12)

New methods of safe biological pest control are required as a result of evolution of insect resistance to current biopesticides. Yeast strains being developed for conversion of cellulosic biomass to ethanol are potential host systems for expression of commercially valuable peptides, such as bioinsecticides, to increase the cost-effectiveness of the process. Spider venom is one of many potential sources of novel insect-specific peptide toxins. Libraries of mutants of the small amphipathic peptide lycotoxin-1 from the wolf spider were produced in high throughput using an automated integrated plasmid-based functional proteomic platform and screened for ability to kill fall armyworms, a significant cause of damage to corn (maize) and other crops in the United States. Using amino acid scanning mutagenesis (AASM) we generated a library of mutagenized lycotoxin-1 open reading frames (ORF) in a novel small ubiquitin-like modifier (SUMO) yeast expression system. The SUMO technology enhanced expression and improved generation of active lycotoxins. The mutants were engineered to be expressed at high level inside the yeast and ingested by the insect before being cleaved to the active form (so-called Trojan horse strategy). These yeast strains expressing mutant toxin ORFs were also carrying the xylose isomerase (XI) gene and were capable of aerobic growth on xylose. Yeast cultures expressing the peptide toxins were prepared and fed to armyworm larvae to identify the mutant toxins with greatest lethality. The most lethal mutations appeared to increase the ability of the toxin alpha-helix to interact with insect cell membranes or to increase its pore-forming ability, leading to cell lysis. The toxin peptides have potential as value-added coproducts to increase the cost-effectiveness of fuel ethanol bioproduction.

The molecular biological techniques for plasmid-based assembly and cloning of gene open reading frames are essential for elucidating the function of the proteins encoded by the genes. High-throughput integrated robotic molecular biology platforms that have the capacity to rapidly clone and express heterologous gene open reading frames in bacteria and yeast and to screen large numbers of expressed proteins for optimized function are an important technology for improving microbial strains Published by Elsevier Inc. on behalf of the Society for Laboratory Automation and Screening for biofuel production. The process involves the production of full-length complementary DNA libraries as a source of plasmid-based clones to express the desired proteins in active form for determination of their functions. Proteins that were identified by high-throughput screening as having desired characteristics are overexpressed in microbes to enable them to perform functions that will allow more cost-effective and sustainable production of biofuels. Because the plasmid libraries are composed of several thousand unique genes, automation of the process is essential. This review describes the design and implementation of an automated integrated programmable robotic workcell capable of producing complementary DNA libraries, colony picking, isolating plasmid DNA, transforming yeast and bacteria, expressing protein, and performing appropriate functional assays. These operations will allow tailoring microbial strains to use renewable feedstocks for production of biofuels, bioderived chemicals, fertilizers, and other coproducts for profitable and sustainable biorefineries.

Background: The field of plasmid-based functional proteomics requires the rapid assay of proteins expressed from plasmid libraries. Automation is essential since large sets of mutant open reading frames are being cloned for evaluation. To date no integrated automated platform is available to carry out the entire process including production of plasmid libraries, expression of cloned genes, and functional testing of expressed proteins.

A yeast artificial chromosome (YAC) containing a multigene cassette for expression of enzymes that enhance xylose utilization (xylose isomerase [XI] and xylulokinase [XKS]) was constructed and transformed into Saccharomyces cerevisiae to demonstrate feasibility as a stable protein expression system in yeast and to design an assembly process suitable for an automated platform. Expression of XI and XKS from the YAC was confirmed by Western blot and PCR analyses. The recombinant and wild-type strains showed similar growth on plates containing hexose sugars, but only recombinant grew on D-xylose and L-arabinose plates. In glucose fermentation, doubling time (4.6 h) and ethanol yield (0.44 g ethanol/g glucose) of recombinant were comparable to wild type (4.9 h and 0.44 g/g). In whole-corn hydrolysate, ethanol yield (0.55 g ethanol/g [glucose + xylose]) and xylose utilization (38%) for recombinant were higher than for wild type (0.47 g/g and 12%). In hydrolysate from spent coffee grounds, yield was 0.46 g ethanol/g (glucose + xylose), and xylose utilization was 93% for recombinant. These results indicate introducing a YAC expressing XI and XKS enhanced xylose utilization without affecting integrity of the host strain, and the process provides a potential platform for automated synthesis of a YAC for expression of multiple optimized genes to improve yeast strains.