A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

Development of CRISPR-Cas9 knock-in tools for free fatty acid production using the fast-growing cyanobacterial strain Synechococcus elongatus UTEX 2973.

Synechococcus elongatus UTEX 2973 has one of the fastest measured doubling time of cyanobacteria making it an important candidate for metabolic engineering. Traditional genetic engineering methods, which rely on homologous recombination, however, are inefficient, labor-intensive, and time-consuming due to the oligoploidy or polyploidy nature of cyanobacteria and the reliance on unique antibiotic resistance markers. CRISPR-Cas9 has emerged as an effective and versatile editing platform in a wide variety of organisms, but its application for cyanobacterial engineering is limited by the inherent toxicity of Cas9 resulting in poor transformation efficiencies. Here, we demonstrated that a single-plasmid CRISPR-Cas9 system, pCRISPOmyces-2, can effectively knock-in a truncated thioesterase gene from Escherichia coli to generate free fatty acid (FFA) producing mutants of Syn2973. To do so, three parameters were evaluated on the effect of generating recipient colonies after conjugation with pCRISPOmyces-2-based plasmids: 1) a modified conjugation protocol termed streaked conjugation, 2) the deletion of the gene encoding RecJ exonuclease, and 3) single guide RNA (sgRNA) sequence. With the use of the streaked conjugation protocol and a ΔrecJ mutant strain of Syn2973, the conjugation efficiency for the pCRISPomyces-2 plasmid could be improved by 750-fold over the wildtype (WT) for a conjugation efficiency of 2.0 × 10-6 transconjugants/recipient cell. While deletion of the RecJ exonuclease alone increased the conjugation efficiency by 150-fold over the WT, FFA generation was impaired in FFA-producing mutants with the ΔrecJ background, and the large number of poor FFA-producing isolates indicated the potential increase in spontaneous mutation rates. The sgRNA sequence was found to be critical in achieving the desired CRISPR-Cas9-mediated knock-in mutation as the sgRNA impacts conjugation efficiency, likelihood of homogenous recombinants, and free fatty acid production in engineered strains.

Occurrence of respiratory viruses on school desks.

BACKGROUND: Schools represent high occupancy environments and well-documented high-risk locations for the transmission of respiratory viruses. The goal of this study was to report on the area density, occurrence, and type of respiratory viruses on desks in primary school classrooms. METHODS: Quantitative reverse transcription polymerase chain reaction (qPCR) techniques were employed to measure nucleic acid area densities from a broad range of human adenoviruses and rhinoviruses, as well as coronavirus OC43, influenza A, and norovirus GI. Every two weeks, virus monitoring was conducted on the desks of four primary school classrooms in Colorado, USA, during the 2019 respiratory virus season. RESULTS: DNA and RNA from respiratory viruses and norovirus were recovered from more than 20% of the desks sampled; occurrence patterns that indicate a greater than 60% probability of encountering any virus, if more than five desks were occupied in a day. Rhinoviruses and adenoviruses were the most commonly detected viruses as judged by the composite of occurrence and number of gene copies recovered. Desktop adenosine triphosphate monitoring did not predict the recovery of viral genomic materials on desks. School desks can be commonly contaminated with respiratory viruses. CONCLUSIONS: Genomic surveys of the identity, distribution and abundance of human viruses on "high-touch" surfaces, can help inform risk assessments, design cleaning interventions, and may be useful for infection surveillance.

Cultivation of marine microalgae using shale gas flowback water and anaerobic digestion effluent as the cultivation medium.

The potential of shale gas flowback water and anaerobic digestion (AD) effluent to reduce the water and nutrient requirements for marine microalgae cultivation was evaluated with the following strains: Nannochloropsis salina, Dunaliella tertiolecta, and Dunaliella salina. N. salina and D. tertiolecta achieved the highest biomass productivity in the medium composed of flowback water and AD effluent (6% v/v). Growth in the above unsterilized medium was found to be comparable to that in sterilized commercial media with similar initial inorganic nitrogen concentrations, salinity, and pH levels. Specific growth rates of 0.293 and 0.349 day(-1) and average biomass productivities of 225 and 275 mg L(-1)day(-1) were obtained for N. salina and D. tertiolecta, respectively. The lipid content and fatty acid profile of both strains in the medium were also comparable to those obtained with commercial nutrients and salts.

Laboratory comparison of four iron-based filter materials for drainage water phosphate treatment.

A laboratory investigation evaluated phosphate (PO4(3-)) drainage water treatment capabilities of four iron-based filter materials. The iron-based filter materials tested were zero-valent iron (ZVI), porous iron composite (PIC), sulfur modified iron (SMI), and iron oxide/ hydroxide (IOH). Only filter material retained on a 60-mesh sieve (> 0.25 mm) was used for evaluation. The laboratory investigation included saturated falling-head hydraulic conductivity tests, contaminant removal or desorption/dissolution batch tests, and low-to-high flow rate saturated solute transport column tests. Each of the four iron-based filter materials have sufficient water flow capacity as indicated by saturated hydraulic conductivity values that in most cases were greater than 1 x 10(-2) cm/s. For the 1, 10, and 100 ppm PO4(3-)-P contaminant removal batch tests, each of the four iron-based filter materials removed at least 95% of the PO4(3-)-P originally present. However, for the 1000 ppm PO4(3-)-P contaminant removal batch tests, IOH by far exhibited the greatest removal effectiveness (99% PO4(3-)-P removal), followed by SMI (72% PO4(3-)-P removal), then ZVI (62% PO4(3-)-P removal), and finally PIC (15% PO4(3-)-P removal). The desorption/dissolution batch test results, especially with respect to SMI and IOH, indicate that once PO4(3-) is adsorbed/precipitated onto surfaces of iron-based filter material particles, this PO4(3-) becomes fixed and is then not readily desorbed/dissolved back into solution. The results from the column tests showed that regardless of low or high flow rate (contact time ranged from a few hours to a few minutes) and PO4(3-) concentration (1 ppm or 10 ppm PO4(3-)-P), PIC, SMI, and IOH reduced PO4(3-)-P concentrations to below detection limits, while ZVI removed at least 90% of the influent PO4(3-)-P. Consequently, these laboratory results indicate that the ZVI, PIC, SMI, and IOH filter materials all exhibit promise for phosphate drainage water treatment.