Genome-scale genetic manipulation methods for exploring bacterial molecular biology

2012 ◽  
Vol 8 (6) ◽  
pp. 1626 ◽  
Author(s):  
Alla Gagarinova ◽  
Andrew Emili
Keyword(s):  
Molecular Biology ◽  
Genetic Manipulation ◽  
Genome Scale

scholarly journals PPM1D is a neuroblastoma oncogene and therapeutic target in childhood neural tumors

2020 ◽  
Author(s):  
Jelena Milosevic ◽  
Susanne Fransson ◽  
Miklos Gulyas ◽  
Gabriel Gallo-Oller ◽  
Thale K Olsen ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Genetic Manipulation ◽  
Clonal Evolution ◽  
Neuroblastoma Cell ◽  
Tumor Formation ◽  
T Cell Lymphomas ◽  
Wip1 Phosphatase ◽  
Bona Fide ◽  
Genome Scale

SUMMARYMajority of cancers harbor alterations of the tumor suppressor TP53. However, childhood cancers, including unfavorable neuroblastoma, often lack TP53 mutations despite frequent loss of p53 function, suggesting alternative p53 inactivating mechanisms.Here we show that p53-regulating PPM1D at chromosome 17q22.3 is linked to aggressive tumors and poor prognosis in neuroblastoma. We identified that WIP1-phosphatase encoded by PPM1D, is activated by frequent segmental 17q-gain further accumulated during clonal evolution, gene-amplifications, gene-fusions or gain-of-function somatic and germline mutations. Pharmacological and genetic manipulation established WIP1 as a druggable target in neuroblastoma. Genome-scale CRISPR-Cas9 screening demonstrated PPM1D genetic dependency in TP53 wild-type neuroblastoma cell lines, and shRNA PPM1D knockdown significantly delayed in vivo tumor formation. Establishing a transgenic mouse model overexpressing PPM1D showed that these mice develop cancers phenotypically and genetically similar to tumors arising in mice with dysfunctional p53 when subjected to low-dose irradiation. Tumors include T-cell lymphomas harboring Notch1-mutations, Pten-deletions and p53-accumulation, adenocarcinomas and PHOX2B-expressing neuroblastomas establishing PPM1D as a bona fide oncogene in wtTP53 cancer and childhood neuroblastoma. Pharmacological inhibition of WIP1 suppressed the growth of neural tumors in nude mice proposing WIP1 as a therapeutic target in neural childhood tumors.

scholarly journals Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Brian M. Luna ◽  
Amber Ulhaq ◽  
Jun Yan ◽  
Paul Pantapalangkoor ◽  
Travis B. Nielsen ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Acinetobacter Baumannii ◽  
Genetic Manipulation ◽  
Multidrug Resistant ◽  
Molecular Techniques ◽  
Drug Resistant ◽  
Selectable Markers ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Selection Of

ABSTRACT Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics. Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

scholarly journals Genome-scale mining of root-preferential genes from maize and characterization of their promoter activity

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Ye Li ◽  
Xiaoqing Liu ◽  
Rumei Chen ◽  
Jian Tian ◽  
Yunliu Fan ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Root Traits ◽  
Crop Productivity ◽  
Data Sets ◽  
Crop Species ◽  
Biotic Stresses ◽  
Maize Cultivars ◽  
Genomic Scale ◽  
Functional Analyses ◽  
Genome Scale

Abstract Background Modification of root architecture and improvement of root resistance to stresses can increase crop productivity. Functional analyses of root-specific genes are necessary for root system improvement, and root-specific promoters enable research into the regulation of root development and genetic manipulation of root traits. Maize is an important crop species; however, little systematic mining of root-specific genes and promoters has been performed to date. Results Genomic-scale mining based on microarray data sets followed by transcript detection resulted in the identification of 222 root-specific genes. Gene Ontology enrichment analyses revealed that these 222 root-specific genes were mainly involved in responses to chemical, biotic, and abiotic stresses. Of the 222 genes, 33 were verified by quantitative reverse transcription polymerase chain reaction, and 31 showed root-preferential activity. About 2 kb upstream 5 of the 31 identified root-preferential genes were cloned from the maize genome as putative promoters and named p8463, p5023, p1534, p8531 and p6629. GUS staining of transgenic maize-derived promoter-GUS constructs revealed that the five promoters drove GUS expression in a root-preferential manner. Conclusions We mined root-preferential genes and their promoters in maize and verified p8463, p5023, p1534, p8531 and p6629 as root-preferential promoters. Our research enables the identification of other tissue-specific genes and promoters in maize and other species. In addition, the five promoters may enable enhancement of target gene(s) of maize in a root-preferential manner to generate novel maize cultivars with resistance to water, fertilizer constraints, or biotic stresses.

scholarly journals Reconstruction of a generic genome-scale metabolic network for chicken: investigating network connectivity and finding potential biomarkers

2021 ◽  
Author(s):  
Ehsan Salehabadi ◽  
Ehsan Motamedian ◽  
Seyed Abbas Shojaosadati
Keyword(s):  
Carbohydrate Metabolism ◽  
Metabolic Pathways ◽  
Egg Production ◽  
Genetic Manipulation ◽  
Human Life ◽  
Principal Component ◽  
Metabolic Model ◽  
Fat Cell ◽  
Chicken Cell ◽  
Genome Scale

Chicken is the first sequenced avian that has a crucial role in human life for its meat and egg production. Because of various metabolic disorders, study the metabolism of chicken cell is important. Herein, the first genome-scale metabolic model of a chicken cell named iES1300, consists of 2427 reactions, 2569 metabolites, and 1300 genes, was reconstructed manually based on databases. Interactions of metabolic genes for growth were examined for E. coli , S. cerevisiae , human, and chicken metabolic models. The results indicated robustness to genetic manipulation for iES1300 similar to the results for human. iES1300 was integrated with transcriptomics data using algorithms and Principal Component Analysis was applied to compare context-specific models of the normal, tumor, lean and fat cell lines. It was found that the normal model has notable metabolic flexibility in the utilization of various metabolic pathways, especially in metabolic pathways of the carbohydrate metabolism, compared to the others. It was also concluded that the fat and tumor models have similar growth metabolisms and the lean chicken model has a more active lipid and carbohydrate metabolism.

scholarly journals A minimal CRISPR-Cas3 system for genome engineering

2019 ◽  
Author(s):  
Bálint Csörgő ◽  
Lina M. León ◽  
Ilea J. Chau-Ly ◽  
Alejandro Vasquez-Rifo ◽  
Joel D. Berry ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Genome Engineering ◽  
High Efficiency ◽  
Genetic Manipulation ◽  
Point Mutations ◽  
Type I ◽  
Homology Directed Repair ◽  
Large Deletions ◽  
System Type ◽  
Genome Scale

AbstractCRISPR-Cas technologies have provided programmable gene editing tools that have revolutionized research. The leading CRISPR-Cas9 and Cas12a enzymes are ideal for programmed genetic manipulation, however, they are limited for genome-scale interventions. Here, we utilized a Cas3-based system featuring a processive nuclease, expressed endogenously or heterologously, for genome engineering purposes. Using an optimized and minimal CRISPR-Cas3 system (Type I-C) programmed with a single crRNA, large deletions ranging from 7 - 424 kb were generated in Pseudomonas aeruginosa with high efficiency and speed. By comparison, Cas9 yielded small deletions and point mutations. Cas3-generated deletion boundaries were variable in the absence of a homology-directed repair (HDR) template, and successfully and efficiently specified when present. The minimal Cas3 system is also portable; large deletions were induced with high efficiency in Pseudomonas syringae and Escherichia coli using an “all-in-one” vector. Notably, Cas3 generated bi-directional deletions originating from the programmed cut site, which was exploited to iteratively reduce a P. aeruginosa genome by 837 kb (13.5%) using 10 distinct crRNAs. We also demonstrate the utility of endogenous Cas3 systems (Type I-C and I-F) and develop an “anti-anti-CRISPR” strategy to circumvent endogenous CRISPR-Cas inhibitor proteins. CRISPR-Cas3 could facilitate rapid strain manipulation for synthetic biological and metabolic engineering purposes, genome minimization, and the analysis of large regions of unknown function.

scholarly journals Molecular Biology of Chili Pepper Anthocyanin Biosynthesis

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
César Aza-González ◽  
Héctor Gordon Núñez-Palenius ◽  
Neftalí Ochoa-Alejo
Keyword(s):  
Molecular Biology ◽  
Biosynthetic Pathway ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Anthocyanin Biosynthesis ◽  
Chili Pepper ◽  
Horticultural Crop ◽  
Research Areas ◽  
Capsicum Spp ◽  
Chili Peppers

Chili pepper (<em>Capsicum spp.</em>) is an important horticultural crop worldwide. Chili pepper fruits from different <em>Capsicum </em>species have been highly consumed in Mexico since pre-Columbian times. Some <em>Capsicum</em> species synthesize and accumulate anthocyanins in different tissues and organs. Although the anthocyanin biosynthetic pathway has been established for different plant species, very few studies on anthocyanin chemistry, biochemistry and molecular biology of these pigments produced by chili peppers have been reported. In this review we describe the information on the type of anthocyanins synthesized and accumulated in chili pepper, and also on the molecular biology of the biosynthetic pathway. Additionally, we discuss the applications of current knowledge for the genetic manipulation, through genetic engineering, of this trait, and also the future anthocyanin-related research areas in <em>Capsicum.</em>

scholarly journals Strategies for Enhancing in vitro Degradation of Linuron by Variovorax sp. Strain SRS 16 Under the Guidance of Metabolic Modeling

2021 ◽  
Vol 9 ◽  
Author(s):  
Kusum Dhakar ◽  
Raphy Zarecki ◽  
Daniella van Bommel ◽  
Nadav Knossow ◽  
Shlomit Medina ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Nitrogen Sources ◽  
Metabolic Model ◽  
Scale Model ◽  
Promoting Effect ◽  
Metabolic Potential ◽  
Carbon And Nitrogen ◽  
A Genome ◽  
Genome Scale

Phenyl urea herbicides are being extensively used for weed control in both agricultural and non-agricultural applications. Linuron is one of the key herbicides in this family and is in wide use. Like other phenyl urea herbicides, it is known to have toxic effects as a result of its persistence in the environment. The natural removal of linuron from the environment is mainly carried through microbial biodegradation. Some microorganisms have been reported to mineralize linuron completely and utilize it as a carbon and nitrogen source. Variovorax sp. strain SRS 16 is one of the known efficient degraders with a recently sequenced genome. The genomic data provide an opportunity to use a genome-scale model for improving biodegradation. The aim of our study is the construction of a genome-scale metabolic model following automatic and manual protocols and its application for improving its metabolic potential through iterative simulations. Applying flux balance analysis (FBA), growth and degradation performances of SRS 16 in different media considering the influence of selected supplements (potential carbon and nitrogen sources) were simulated. Outcomes are predictions for the suitable media modification, allowing faster degradation of linuron by SRS 16. Seven metabolites were selected for in vitro validation of the predictions through laboratory experiments confirming the degradation-promoting effect of specific amino acids (glutamine and asparagine) on linuron degradation and SRS 16 growth. Overall, simulations are shown to be efficient in predicting the degradation potential of SRS 16 in the presence of specific supplements. The generated information contributes to the understanding of the biochemistry of linuron degradation and can be further utilized for the development of new cleanup solutions without any genetic manipulation.

Sign in / Sign up

Export Citation Format

Share Document