Genome Sequence of Escherichia coli Stbl4, a Versatile Genetic Tool for Heterologous Expression

Escherichia coli Stbl4 is widely used as a laboratory strain for heterologous expression of large gene clusters. Since no genome sequence has been publicly available, we here report the draft sequence of Stbl4, including its F-plasmid. It should serve as a useful reference for researchers working with Stbl4.

A Novel Cre/lox-Based Genetic Tool for Repeated, Targeted and Markerless Gene Integration in Yarrowia lipolytica

The unconventional yeast Yarrowia lipolytica is extensively applied in bioproduction fields owing to its excellent metabolite and protein production ability. Nonetheless, utilization of this promising host is still restricted by the limited availability of precise and effective gene integration tools. In this study, a novel and efficient genetic tool was developed for targeted, repeated, and markerless gene integration based on Cre/lox site-specific recombination system. The developed tool required only a single selection marker and could completely excise the unnecessary sequences. A total of three plasmids were created and seven rounds of marker-free gene integration were examined in Y. lipolytica. All the integration efficiencies remained above 90%, and analysis of the protein production and growth characteristics of the engineered strains confirmed that genome modification via the novel genetic tool was feasible. Further work also confirmed that the genetic tool was effective for the integration of other genes, loci, and strains. Thus, this study significantly promotes the application of the Cre/lox system and presents a powerful tool for genome engineering in Y. lipolytica.

CRISPR-PN2: a flexible and genome-aware platform for diverse CRISPR experiments in parasitic nematodes

Parasitic nematodes represent a significant threat to human health, causing diseases of major socioeconomic importance worldwide. Central to controlling infections of parasitic nematodes is a more detailed molecular picture of host specificity, parasite activation and immune suppression. CRISPR technology holds huge potential for researchers in the field of parasitic nematology, as it provides a powerful genetic tool to dissect questions in parasite biology. To expedite the development of CRISPR technology in parasitic nematodes, software is required to facilitate the design of effective and specific sgRNA sequences. Here, the author introduces CRISPR-PN2, a comprehensive web-based platform that provides flexible use control over the automated design of specific gRNA sequences for CRISPR experiments in parasitic nematodes.

Application of the Mineralogy and Mineral Chemistry of Carbonates as a Genetic Tool in the Hydrothermal Environment

The mineralogy and mineral chemistry of carbonates from various hydrothermal deposits, including volcanic-hosted Au-Cu epithermal, “Chilean Manto-type” Cu(-Ag), stratabound Mn, and Ag-Ba vein deposits from Spain and Chile, were investigated. Dolomite-ankerite (±siderite) was found in variable amounts within the epithermal deposits and associated hydrothermal alteration, whereas calcite was found either within barren veins or disseminated within the regional alteration. Calcite is the major gangue phase within the stratabound deposits, which tend to lack dolomite/ankerite and siderite. Carbonates precipitated from hydrothermal ore fluids are typically Mn-rich, up to 3.55 at. % in siderite, 2.27 at. % in dolomite/ankerite, and 1.92 at. % in calcite. In contrast, calcite related to very low-grade metamorphism or regional low-temperature alteration is Mn-poor but sometimes Mg-rich, possibly related to a higher temperature of formation. Chemical zonation was observed in the hydrothermal carbonates, although no unique pattern and chemical evolution was observed. This study suggests that the chemical composition of carbonates, especially the Mn content, could be a useful vector within ore-forming hydrothermal systems, and therefore constitutes a possible tool in geochemical exploration. Furthermore, Mn-poor calcites detected in some deposits are suggested to be linked with a later episode, maybe suggesting a predominance of meteoric waters, being not related to the main ore stage formation, thus avoiding misunderstanding of further isotopic studies.

Missense Variants of Uncertain Significance: A Powerful Genetic Tool for Function Discovery with Clinical Implications

The breast cancer susceptibility gene BRCA2 encodes a multifunctional protein required for the accurate repair of DNA double-strand breaks and replicative DNA lesions. In addition, BRCA2 exhibits emerging important roles in mitosis. As a result, mutations in BRCA2 may affect chromosomal integrity in multiple ways. However, many of the BRCA2 mutations found in breast cancer patients and their families are single amino acid substitutions, sometimes unique, and their relevance in cancer risk remains difficult to assess. In this review, we focus on three recent reports that investigated variants of uncertain significance (VUS) located in the N-terminal region of BRCA2. In this framework, we make the case for how the functional evaluation of VUS can be a powerful genetic tool not only for revealing novel aspects of BRCA2 function but also for re-evaluating cancer risk. We argue that other functions beyond homologous recombination deficiency or “BRCAness” may influence cancer risk. We hope our discussion will help the reader appreciate the potential of these functional studies in the prevention and diagnostics of inherited breast and ovarian cancer. Moreover, these novel aspects in BRCA2 function might help find new therapeutic strategies.

Optimizing Transformation Frequency of Cryptococcus neoformans and Cryptococcus gattii Using Agrobacterium tumefaciens

The transformation of Cryptococcus spp. by Agrobacterium tumefaciens has proven to be a useful genetic tool. A number of factors affect transformation frequency. These factors include acetosyringone concentration, bacterial cell to yeast cell ratio, cell wall damage, and agar concentration. Agar concentration was found to have a significant effect on the transformant number as transformants increased with agar concentration across all four serotypes. When infection time points were tested, higher agar concentrations were found to result in an earlier transfer of the Ti-plasmid to the yeast cell, with the earliest transformant appearing two h after A. tumefaciens contact with yeast cells. These results demonstrate that A. tumefaciens transformation efficiency can be affected by a variety of factors and continued investigation of these factors can lead to improvements in specific A. tumefaciens/fungus transformation systems.

Isolation of Mutants With Reduced Susceptibility to Piperaquine From a Mutator of the Rodent Malaria Parasite Plasmodium berghei

Elucidation of the mechanisms of drug resistance in malaria parasites is crucial for combatting the emergence and spread of resistant parasites, which can be achieved by tracing resistance-associated mutations and providing useful information for drug development. Previously, we produced a novel genetic tool, a Plasmodium berghei mutator (PbMut), whose base substitution rate is 36.5 times higher than that of wild-type parasites. Here, we report the isolation of a mutant with reduced susceptibility to piperaquine (PPQ) from PbMut under PPQ pressure by sequential nine-cycle screening and named it PbMut-PPQ-R-P9. The ED50 of PbMut-PPQ-R-P9 was 1.79 times higher than that of wild-type parasites, suggesting that its PPQ resistance is weak. In the 1st screen, recrudescence occurred in the mice infected with PbMut but not in those infected with wild-type parasites, suggesting earlier emergence of PPQ-resistant parasites from PbMut. Whole-genome sequence analysis of PbMut-PPQ-R-P9 clones revealed that eight nonsynonymous mutations were conserved in all clones, including N331I in PbCRT, the gene encoding chloroquine resistance transporter (CRT). The PbCRT(N331I) mutation already existed in the parasite population after the 2nd screen and was predominant in the population after the 8th screen. An artificially inserted PbCRT(N331I) mutation gave rise to reduced PPQ susceptibility in genome-edited parasites (PbCRT-N331I). The PPQ susceptibility and growth rates of PbCRT-N331I parasites were significantly lower than those of PbMut-PPQ-R-P9, implying that additional mutations in the PbMut-PPQ-R9 parasites could compensate for the fitness cost of the PbCRT(N331I) mutation and contribute to reduced PPQ susceptibility. In summary, PbMut could serve as a novel genetic tool for predicting gene mutations responsible for drug resistance. Further study on PbMut-PPQ-R-P9 could identify genetic changes that compensate for fitness costs owing to drug resistance acquisition.

The Tn7-based genomic integration is dependent on an attTn7 box in the glms gene and is site-specific with monocopy in Ralstonia solanacearum species complex

The Tn7-based genomic integration system enables direct insertion of foreign gene elements into chromosome downstream of glms in many bacteria species. The glms gene is greatly conserved in Ralstonia solanacearum species complex (RSSC), while its downstream regions are mostly different in the RSSC. Here, we provided genetic evidence to validate that this Tn7-integration is dependent on a conserved 30-bp motif in the glms, called attTn7 box, and artificial attTn7 boxes elsewhere are competent for the Tn7-integration, which is further confirmed to be simultaneous at downstream of both original and artificial attTn7 boxes using the PCR. With the whole genome re-sequencing on 500 Tn7-colonies, the Tn7-integration was confirmed to be site- specific at 25-bp downstream of glms with monocopy as chromosome of the RSSC. Characteristic of the monocopy in chromosome enables the Tn7-based complementation to fully restore phenotypes of mutants to those of parent strains that is advantageous than those based on plasmids with low-copy numbers. The Tn7-based genomic integration system provides a generally applicable and versatile genetic tool for studies of complementation, pathogenesis, overexpression, and in-vivo promoter activity assays with monocopy in the RSSC.