To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning.

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Phage display is a powerful technology for selecting and engineering peptides and proteins expressed on the surface of filamentous bacteriophage. The advantages of phage display technology over other research tools and its' great potential have been demonstrated by successful application of phage display in diverse fields of biomedical/clinical research. In this review we will describe some recent developments in phage display, including new expression vectors, display formats, bioselection strategies and applications in pharmaceutical biotechnology. We highlight some important applications of phage display to identify disease- and pathogen-specific biomolecules, making particular emphasis on development of phage display-derived preventive and therapeutic vaccines.

Burkholderia are highly evolved Gram-negative bacteria that primarily infect solipeds but are transmitted to humans by ingestion and cutaneous or aerosol exposures. Heightened concern over human infections of Burkholderia mallei and the very closely related species B. pseudomallei is due to the pathogens’ proven effectiveness as bioweapons, and to the increased potential for natural opportunistic infections in the growing diabetic and immuno-compromised populations. These Burkholderia species are nearly impervious to antibiotic treatments and no vaccine exists. In this study, the genome of the highly virulent B. mallei ATCC23344 strain was examined by expression library immunization for gene-encoded protective antigens. This protocol for genomic-scale functional screening was customized to accommodate the unusually large complexity of Burkholderia, and yielded 12 new putative vaccine candidates. Five of the candidates were individually tested as protein immunogens and three were found to confer significant partial protection against a lethal pulmonary infection in a murine model of disease. Determinations of peripheral blood cytokine and chemokine profiles following individual protein immunizations show that interleukin-2 (IL-2) and IL-4 are elicited by the three confirmed candidates, but unexpectedly interferon-γ and tumor necrosis factor-α are not. We suggest that these pathogen components, discovered using genetic immunization and confirmed in a conventional protein format, will be useful toward the development of a safe and effective glanders vaccine.

Neutral electrolyzed water (NEW) was tested as a disinfectant against Listeria monocytogenes on the surface of table eggs. Eggs were collected from a single Bovans White flock and were exposed to L. monocytogenes . Artificially contaminated eggs were divided into three different treatment groups: NEW, 2% citric acid solution (CAS), and saline solution (SS). To evaluate the bactericidal effect, the Mexican norm for antimicrobial activity determination protocol was performed. The observed bactericidal effect was compared against those obtained from CAS and SS. Bacterial cells present on the eggshells were quantified. NEW exhibited a significantly higher bactericidal effect than CAS when evaluated on the surfaces of chicken eggshells (6.11 log 10 CFU/ml reduction in vitro and a 2.18 log 10 CFU/egg reduction on eggs vs. 1.06 log 10 CFU/ml in vitro reduction and 1.74 log 10 CFU/egg). Additionally, CAS was found to react with the carbonate egg shield, resulting in a loss of cuticle integrity. Mineral content of NEW‐treated eggshells was similar to SS‐treated eggshells; however, CAS‐treated eggshells showed a significant decrease in phosphorous concentration compared to NEW treatment. In this study, we demonstrated the effect of NEW and CAS on the integrity of the L. monocytogenes wall using transmission electron microscopy. To the best of our knowledge, this is the first report of the effect of NEW against L. monocytogenes on eggshells. Our results show that NEW is a viable alternative solution for the disinfection of table eggs that does not affect the cuticle or shell.

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The licensed smallpox vaccine, comprised of infectious vaccinia, is no longer popular as it is associated with a variety of adverse events. Safer vaccines have been explored such as further attenuated viruses and component designs. However, these alternatives typically provide compromised breadth and strength of protection. We conducted a genome-level screening of cowpox, the ancestral poxvirus, in the broadly immune-presenting C57BL/6 mouse as an approach to discovering novel components with protective capacities. Cowpox coding sequences were synthetically built and directly assayed by genetic immunization for open-reading-frames that protect against lethal pulmonary infection. Membrane and non-membrane antigens were identified that partially protect C57BL/6 mice against cowpox and vaccinia challenges without adjuvant or regimen optimization, whereas the 4-pox vaccine did not. New vaccines might be developed from productive combinations of these new and existing antigens to confer potent, broadly-efficacious protection and be contraindicated for none.

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