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Advanced Topics in Molecular Biology
Published in Jay L. Nadeau, Introduction to Experimental Biophysics, 2017
A hybrid of phage and plasmid technology is the cosmid, which is a plasmid containing phage cos sites so that it can concatemerize to form libraries (Figure 4.4). Its cloning capacity is three to five times greater than that of a plasmid, since it does not include any of the arm DNA that leads to the production of viral particles; the entire phage genome space can be taken up by the insert. Replication incompetent helper phage, which are essentially viral empty shells, are used to package the concatemer DNA and infect E. coli. This DNA then replicates in the bacteria like a plasmid. No viral particles are produced, and no plaques form. This type of vector is excellent for large inserts (30–40 kb).
Selection and Improvement of Industrial Organisms for Biotechnological Applications
Published in Nduka Okafor, Benedict C. Okeke, Modern Industrial Microbiology and Biotechnology, 2017
Nduka Okafor, Benedict C. Okeke
Cosmids are plasmids constructed from phage DNA by circularization at the ‘sticky,’ single stranded ends or cos sites. Foreign DNA is attached to the cosmid which is then packaged into a phage. When the cosmid is injected, it circularizes like other virus DNA, but it does not behave as a phage, rather it replicates like a plasmid. Drug resistance markers carried on it help to identify it.
Functional Metagenomics
Published in Vineet Kumar, Vinod Kumar Garg, Sunil Kumar, Jayanta Kumar Biswas, Omics for Environmental Engineering and Microbiology Systems, 2023
Kripa Pancholi, Anupama Shrivastav
The identification of a particular gene depends on many factors such as the size of the target gene and gene expression availability of metagenome linked with one another, and the screening of such gene libraries is summarized further. Here, we present an overview of screening and show the technical issue that leads to the hit rate problem. One of the advanced issues is the partial expression of foreign genes into E. coli. Generally, the common surrogate host is E. coli for forming the libraries and so the gene deficiency occurs for recombination and restriction that help in cloning different or modified DNA into E. coli (Rizzo and Giudice 2020). Selecting the vector highly depends on the length of inserts, and for this, many types of strains are available as highly competent cells. For smaller fragments, say for example 10 kb, plasmids are used, and for larger fragments, cosmids or fosmids are used independently of the length of inserts, which shows that it depends on the size of clones. Among all the vectors, the plasmids have the strongest borne promoters and high copy number, although they do not improve the hit rate (the number of positives per total number of clones screened) significantly. For example, plasmid and fosmid vectors both clone the same enzyme from the same metagenome, but hit rates were quite similar between both procedures (Tansirichaiya et al. 2021) although the plasmid vector can be orientated twice for increasing the chances of transcribing the clone-inserted fragment which also succeeds the increase in the hit rate. The provenance of the metagenome has an impact on the hit rate. The target gene in the ecosystem is enriched through natural or artificial contamination (Bhatt et al. 2021). The enzymatic activities can be known through agar plates where positive clones are identified through clear zone through visual screening. But when the metagenomic libraries are cultivated on plates, then the screening can be identified through colour. Enzyme activities are usually expanded with substrates. There can be a low hit rate for a common reason of imperceptible signals, although the screening can be done without using a special device. The other substitutional approaches such as cell lysates for screening, where the library of cells is grown in 96-well plates having lysates formed by the chemical or physical procedure (Kumar et al. 2020), help in improving the sensitivity. The survival of the host is linked to target activities for obtaining high throughput; this method is used for toxic compounds such as antibiotics or heavy metals (Xing et al. 2020), which helps in the screening of resistance genes. Some hosts lack the target gene, and for identifying and selecting the essential gene of such host, this method can be helpful. And another approach beneficial is reporter assay. Green fluorescent protein, β-galactosidase (Dhanjal et al. 2020), and tetracycline resistance gene (Berglund et al. 2020) are sensitive reporter genes expressed when the biological event is attached to the reporter gene (Figure 3.1).
The application of molecular tools to study the drinking water microbiome – Current understanding and future needs
Published in Critical Reviews in Environmental Science and Technology, 2019
While omics tools have been applied to various microbial ecosystems by many studies, application to the drinking water microbiome is limited to a few studies. Some of these studies are based on the use of cosmid library construction that is low in sequence throughput [212] or early NGS technologies that cannot derive long assembled contigs to provide correct linkage between microbes and functionalities (Chistoserdova, 2014; Schmeisser et al., 2003). Using early NGS techniques, two studies (Chao et al., 2013; Gomez-Alvarez et al., 2012) investigated the impact of water treatment on the drinking water microbiome. Their results revealed that chlorine and chloramine treatments caused differences in community structures, disinfectant mechanisms, and virulence genes (Gomez-Alvarez et al., 2012). Changes in protective functions (i.e., glutathione synthesis) were observed in treated water compared with raw water (Chao et al., 2013). In contrast, the use of the latest NGS technology has provided in-depth information into the metabolic and geochemical potential of groundwater-fed RSFs (Palomo et al., 2016). Dominance of Nitrospira was observed to co-occur with high abundance of genes in nitrification and carbon fixation pathways. Genomic analysis revealed that the Nitrospira genome harbored complete ammonia monooxygenase (amoCAB), particularly, the atypical amoA gene similar to the complete ammonia oxidation bacteria Nitrospira (i.e., comammox). This novel finding suggested that Nitrospira in RSF had the potential for complete ammonium oxidation. Moreover, other recovered draft genomes had the capability to oxidize ammonium, nitrite, hydrogen sulfide, methane, and potentially iron and manganese, as well as to assimilate organic compounds. Similarly, Pinto et al. (2016) reported metagenomic evidence for the presence of a comammox Nitrospira genome in biologically active filters. The recovered bin from metagenomes contained the full suite of ammonia oxidation genes on a single scaffold. Genome-resolved metagenomics could also be used to differentiate pathogens and closely related species and identify new biomarkers, such as spacers located in the clustered regularly interspaced short palindromic repeats (CRISPR) regions for the monitoring of ‘true’ pathogenic strains across different drinking water systems (Zhang, Kitajima, Whittle, & Liu, 2017).