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Cell-free transcription-translation (TXTL) enables achieving an ever-growing number of applications, ranging from the rapid characterization of DNA parts to the production of biologics. As TXTL systems gain in versatility and efficacy, larger DNAs can be expressed in vitro extending the scope of cell-free biomanufacturing to new territories. The demonstration that complex entities such as infectious bacteriophages can be synthesized from their genomes in TXTL reactions opens new opportunities, especially for biomedical applications. Over the last century, phages have been instrumental in the discovery of many ground-breaking biotechnologies including CRISPR. The primary function of phages is to infect bacteria. In that capacity, phages are considered an alternative approach to tackling current societal problems such as the rise of antibiotic-resistant microbes. TXTL provides alternative means to produce phages and with several advantages over in vivo synthesis methods. In this chapter, we describe the basic procedures to purify phage genomes, cell-free synthesize phages, and quantitate them using an all-E. coli TXTL system. 

Abstract Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 10¹¹PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome. 

Wildfires have continued to increase in frequency and severity in Southern California due in part to climate change. To gain a further understanding of microbial soil communities’ response to fire and functions that may enhance post-wildfire resilience, soil fungal and bacterial microbiomes were studied from different wildfire areas in the Gold Creek Preserve within the Angeles National Forest using 16S, FITS, 18S, 12S, PITS, and COI amplicon sequencing. Sequencing datasets from December 2020 and June 2021 samplings were analyzed using QIIME2, ranacapa, stats, vcd, EZBioCloud, and mixomics. Significant differences were found among bacterial and fungal taxa associated with different fire areas in the Gold Creek Preserve. There was evidence of seasonal shifts in the alpha diversity of the bacterial communities. In the sparse partial least squares analysis, there were strong associations (r > 0.8) between longitude, elevation, and a defined cluster of Amplicon Sequence Variants (ASVs). The Chi-square test revealed differences in fungi–bacteria (F:B) proportions between different trails (p = 2 × 10−16). sPLS results focused on a cluster of Green Trail samples with high elevation and longitude. Analysis revealed the cluster included the post-fire pioneer fungi Pyronema and Tremella. Chlorellales algae and possibly pathogenic Fusarium sequences were elevated. Bacterivorous Corallococcus, which secretes antimicrobials, and bacterivorous flagellate Spumella were associated with the cluster. There was functional redundancy in clusters that were differently composed but shared similar ecological functions. These results implied a set of traits for post-fire resiliency. These included photo-autotrophy, mineralization of pyrolyzed organic matter and aromatic/oily compounds, potential pathogenicity and parasitism, antimicrobials, and N-metabolism.