Simple, Robust Invertebrate DNA Barcoding: Chelex-Based DNA Extraction and Optimized COI Amplification.

Chelex-based DNA extractions are well suited for student DNA barcoding research because they are simple, safe, and inexpensive and can be performed without specialized laboratory equipment, allowing them to be performed in classrooms or at home. Extracted DNA is stable in Chelex solution for at least a week at ambient temperature, allowing collection of DNA samples from remote students. These extractions provide quality DNA for many taxa and are optimal for barcoding invertebrates, especially in combination with novel cytochrome c oxidase I (COI) primer cocktails and PCR cycling conditions.

Cost-Effective DNA Extraction for DNA Barcoding Diverse Biological Samples.

DNA barcoding employs standard molecular techniques (e.g., DNA extraction, PCR, and Sanger sequencing) to taxonomically identify biological samples. While DNA barcoding is a useful experimental workflow for in-class active learning exercises, extracting DNA from diverse sample types in a time and cost-effective manner can be challenging in a classroom setting. Here, we provide two time and cost-effective methods that have been used by novice students to successfully extract DNA from a variety of animal, fungal, algal, and plant tissues for DNA barcoding.

A Rapid, Equipment-Free DNA Isolation Method for DNA Barcoding.

This rapid, equipment-free DNA isolation procedure using chromatography paper is a simple method that can be performed in less than 30 min and requires no wet lab experience. With minimal expense, it offers an affordable alternative for anyone wanting to explore biodiversity. It also provides an excellent option for use in classrooms or other activities that are time limited. The method works best for plants or lichens, producing stable DNA on Whatman® chromatography paper at room temperature, which can be eluted as needed.

Analysis of DNA Barcodes Using DNA Subway.

DNA Subway makes bioinformatic analysis of DNA barcodes classroom friendly, eliminating the need for software installations or command line tools. Subway bundles research-grade bioinformatics software into workflows with an easy-to-use interface. This chapter covers DNA Subway's DNA barcoding analysis workflow (Blue Line) starting with one or more Sanger sequence reads. During analysis, users can view trace files and sequence quality, pair and align forward and reverse reads, create and trim consensus sequences, perform BLAST searches, select reference data, align multiple sequences, and compute phylogenetic trees. High-quality sequences with the required metadata can also be submitted as barcode sequences to NCBI GenBank.

Cell-Free Synthesis and Quantitation of Bacteriophages.

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.

PHEIGES: all-cell-free phage synthesis and selection from engineered genomes.

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 1011 PFU/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.

Assessing microbial diversity in soil samples along the Potomac River: implications for environmental health.

IMPORTANCE: This study integrates microbial analysis into an undergraduate chemistry class, offering students a hands-on approach to environmental research. We examined the soil along the urbanized Potomac River, discovering a mix of common marine microbes and others that are indicators of urban waste and pollution. Our findings provide valuable insights into the environmental impacts of urbanization on soil health and reveal the effectiveness of using modern genetic tools to teach students about real-world issues. This innovative educational approach not only deepens students' understanding of chemistry and ecology but also prepares them to be thoughtful, informed participants in addressing contemporary environmental challenges while shedding light on the state of the soil microbiome near and around the DC metro area.

The Functional Biogeography of eDNA Metacommunities in the Post-Fire Landscape of the Angeles National Forest.

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.

DNA barcoding Brooklyn (New York): A first assessment of biodiversity in Marine Park by citizen scientists.

DNA barcoding is both an important research and science education tool. The technique allows for quick and accurate species identification using only minimal amounts of tissue samples taken from any organism at any developmental phase. DNA barcoding has many practical applications including furthering the study of taxonomy and monitoring biodiversity. In addition to these uses, DNA barcoding is a powerful tool to empower, engage, and educate students in the scientific method while conducting productive and creative research. The study presented here provides the first assessment of Marine Park (Brooklyn, New York, USA) biodiversity using DNA barcoding. New York City citizen scientists (high school students and their teachers) were trained to identify species using DNA barcoding during a two-week long institute. By performing NCBI GenBank BLAST searches, students taxonomically identified 187 samples (1 fungus, 70 animals and 116 plants) and also published 12 novel DNA barcodes on GenBank. Students also identified 7 ant species and demonstrated the potential of DNA barcoding for identification of this especially diverse group when coupled with traditional taxonomy using morphology. Here we outline how DNA barcoding allows citizen scientists to make preliminary taxonomic identifications and contribute to modern biodiversity research.

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes.

To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.