scholarly journals The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae

Metabolites ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 49 ◽  
Author(s):  
Ana Molina-Márquez ◽  
Marta Vila ◽  
Javier Vigara ◽  
Ana Borrero ◽  
Rosa León
Keyword(s):  
Genetic Transformation ◽  
Selectable Marker ◽  
Genetic Manipulation ◽  
Plant Cells ◽  
Phytoene Desaturase ◽  
Carotenoid Content ◽  
Great Promise ◽  
Encoding Gene ◽  
Average Transformation ◽  
Selection Of

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

scholarly journals Efficient Targeted Mutagenesis inBorrelia burgdorferi

2000 ◽  
Vol 182 (9) ◽  
pp. 2445-2452 ◽  
Author(s):  
James L. Bono ◽  
Abdallah F. Elias ◽  
John J. Kupko ◽  
Brian Stevenson ◽  
Kit Tilly ◽  
...  
Keyword(s):  
Selectable Marker ◽  
Genetic Manipulation ◽  
Kanamycin Resistance ◽  
Targeted Mutagenesis ◽  
Direct Selection ◽  
Genetic Studies ◽  
B Subunit ◽  
Native Promoter ◽  
Selection Of ◽  
Striking Contrast

ABSTRACT Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrBr , a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumermycin A1. The utility of the coumermycin-resistantgyrBr gene for targeted gene disruption is limited by a high frequency of recombination with the endogenousgyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with thekan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.

scholarly journals Complementation of Arginine Auxotrophy for Genetic Transformation of Coxiella burnetii by Use of a Defined Axenic Medium

2016 ◽  
Vol 82 (10) ◽  
pp. 3042-3051 ◽  
Author(s):  
Kelsi M. Sandoz ◽  
Paul A. Beare ◽  
Diane C. Cockrell ◽  
Robert A. Heinzen
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Transformation ◽  
Host Cell ◽  
Coxiella Burnetii ◽  
Genetic Manipulation ◽  
Defined Medium ◽  
Gradual Transition ◽  
Content Type ◽  
Selection Of

ABSTRACTHost cell-free (axenic) culture ofCoxiella burnetiiin acidified citrate cysteine medium-2 (ACCM-2) has provided important opportunities for investigating the biology of this naturally obligate intracellular pathogen and enabled the development of tools for genetic manipulation. However, ACCM-2 has complex nutrient sources that preclude a detailed study of nutritional factors required forC. burnetiigrowth. Metabolic reconstruction ofC. burnetiipredicts that the bacterium cannot synthesize all amino acids and therefore must sequester some from the host. To examineC. burnetiiamino acid auxotrophies, we developed a nutritionally defined medium with known amino acid concentrations, termed ACCM-D. Compared to ACCM-2, ACCM-D supported longer logarithmic growth, a more gradual transition to stationary phase, and approximately 5- to 10-fold greater overall replication. Small-cell-variant morphological forms generated in ACCM-D also showed increased viability relative to that generated in ACCM-2. Lack of growth in amino acid-deficient formulations of ACCM-D revealedC. burnetiiauxotrophy for 11 amino acids, including arginine. Heterologous expression ofLegionella pneumophilaargGHinC. burnetiipermitted growth in ACCM-D missing arginine and supplemented with citrulline, thereby providing a nonantibiotic means of selection ofC. burnetiigenetic transformants. Consistent with bioinformatic predictions, the elimination of glucose did not impairC. burnetiireplication. Together, these results highlight the advantages of a nutritionally defined medium in investigations ofC. burnetiimetabolism and the development of genetic tools.IMPORTANCEHost cell-free growth and genetic manipulation ofCoxiella burnetiihave revolutionized research of this intracellular bacterial pathogen. Nonetheless, undefined components of growth medium have made studies ofC. burnetiiphysiology difficult and have precluded the development of selectable markers for genetic transformation based on nutritional deficiencies. Here, we describe a medium, containing only amino acids as the sole source of carbon and energy, which supports robust growth and improved viability ofC. burnetii. Growth studies confirmed thatC. burnetiicannot replicate in medium lacking arginine. However, genetic transformation of the bacterium with constructs containing the last two genes in theL. pneumophilaarginine biosynthesis pathway (argGH) allowed growth on defined medium missing arginine but supplemented with the arginine precursor citrulline. Our results advance the field by facilitating studies ofC. burnetiimetabolism and allowing non-antibiotic-based selection ofC. burnetiigenetic transformants, an important achievement considering that selectable makers based on antibiotic resistance are limited.

scholarly journals Establishment of a Genetic Transformation System in Guanophilic Fungus Amphichorda guana

2021 ◽  
Vol 7 (2) ◽  
pp. 138
Author(s):  
Min Liang ◽  
Wei Li ◽  
Landa Qi ◽  
Guocan Chen ◽  
Lei Cai ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Genetic Manipulation ◽  
Major Facilitator Superfamily ◽  
Protoplast Regeneration ◽  
Transformation System ◽  
Regeneration Rate ◽  
Bioactive Natural Products ◽  
Genetics Research ◽  
Genetic Transformation System ◽  
Major Facilitator

Fungi from unique environments exhibit special physiological characters and plenty of bioactive natural products. However, the recalcitrant genetics or poor transformation efficiencies prevent scientists from systematically studying molecular biological mechanisms and exploiting their metabolites. In this study, we targeted a guanophilic fungus Amphichorda guana LC5815 and developed a genetic transformation system. We firstly established an efficient protoplast preparing method by conditional optimization of sporulation and protoplast regeneration. The regeneration rate of the protoplast is up to about 34.6% with 0.8 M sucrose as the osmotic pressure stabilizer. To develop the genetic transformation, we used the polyethylene glycol-mediated protoplast transformation, and the testing gene AG04914 encoding a major facilitator superfamily transporter was deleted in strain LC5815, which proves the feasibility of this genetic manipulation system. Furthermore, a uridine/uracil auxotrophic strain was created by using a positive screening protocol with 5-fluoroorotic acid as a selective reagent. Finally, the genetic transformation system was successfully established in the guanophilic fungus strain LC5815, which lays the foundation for the molecular genetics research and will facilitate the exploitation of bioactive secondary metabolites in fungi.

scholarly journals Heterogeneity of mesenchymal stem cells in cartilage regeneration: from characterization to application

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Kangkang Zha ◽  
Xu Li ◽  
Zhen Yang ◽  
Guangzhao Tian ◽  
Zhiqiang Sun ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Articular Cartilage ◽  
Cartilage Repair ◽  
Cartilage Damage ◽  
Cartilage Regeneration ◽  
Great Promise ◽  
Traditional Treatment ◽  
Different Types ◽  
Selection Of

AbstractArticular cartilage is susceptible to damage but hard to self-repair due to its avascular nature. Traditional treatment methods are not able to produce satisfactory effects. Mesenchymal stem cells (MSCs) have shown great promise in cartilage repair. However, the therapeutic effect of MSCs is often unstable partly due to their heterogeneity. Understanding the heterogeneity of MSCs and the potential of different types of MSCs for cartilage regeneration will facilitate the selection of superior MSCs for treating cartilage damage. This review provides an overview of the heterogeneity of MSCs at the donor, tissue source and cell immunophenotype levels, including their cytological properties, such as their ability for proliferation, chondrogenic differentiation and immunoregulation, as well as their current applications in cartilage regeneration. This information will improve the precision of MSC-based therapeutic strategies, thus maximizing the efficiency of articular cartilage repair.

Silenced Phytoene desaturase Gene as a Scorable Marker for Plant Genetic Transformation

2014 ◽  
Vol 13 (2) ◽  
pp. 80-84
Author(s):  
Vikas Yadav Patade ◽  
Deepti Khatri ◽  
Atul Grover ◽  
Maya Kumari ◽  
Sanjay Mohan Gupta ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Phytoene Desaturase ◽  
Desaturase Gene

scholarly journals Ultrasensitive Label-Free Detection of Unamplified Multidrug-Resistance Bacteria Genes with a Bimodal Waveguide Interferometric Biosensor

Diagnostics ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 845
Author(s):  
Jesús Maldonado ◽  
Ana Belén González-Guerrero ◽  
Adrián Fernández-Gavela ◽  
Juan José González-López ◽  
Laura M. Lechuga
Keyword(s):  
Antimicrobial Resistance ◽  
Negative Impact ◽  
Multidrug Resistant ◽  
Therapeutic Interventions ◽  
Great Promise ◽  
Resistant Bacteria ◽  
Label Free ◽  
Multidrug Resistant Bacteria ◽  
Label Free Detection ◽  
Encoding Gene

Infections by multidrug-resistant bacteria are becoming a major healthcare emergence with millions of reported cases every year and an increasing incidence of deaths. An advanced diagnostic platform able to directly detect and identify antimicrobial resistance in a faster way than conventional techniques could help in the adoption of early and accurate therapeutic interventions, limiting the actual negative impact on patient outcomes. With this objective, we have developed a new biosensor methodology using an ultrasensitive nanophotonic bimodal waveguide interferometer (BiMW), which allows a rapid and direct detection, without amplification, of two prevalent and clinically relevant Gram-negative antimicrobial resistance encoding sequences: the extended-spectrum betalactamase-encoding gene blaCTX-M-15 and the carbapenemase-encoding gene blaNDM-5 We demonstrate the extreme sensitivity and specificity of our biosensor methodology for the detection of both gene sequences. Our results show that the BiMW biosensor can be employed as an ultrasensitive (attomolar level) and specific diagnostic tool for rapidly (less than 30 min) identifying drug resistance. The BiMW nanobiosensor holds great promise as a powerful tool for the control and management of healthcare-associated infections by multidrug-resistant bacteria.

scholarly journals Genetic transformation of Coniochaeta sp. 2T2.1, key fungal member of a lignocellulose-degrading microbial consortium

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Nancy N Nichols ◽  
Ronald E Hector ◽  
Sarah E Frazer
Keyword(s):  
Green Fluorescent Protein ◽  
Genetic Transformation ◽  
Pure Culture ◽  
Ecological Niche ◽  
Fluorescent Protein ◽  
Microbial Consortium ◽  
Transformed Cells ◽  
Antibiotic Selection ◽  
Green Fluorescent ◽  
Selection Of

Abstract Coniochaeta sp. strain 2T2.1 is a key member of a microbial consortium that degrades lignocellulosic biomass. Due to its ecological niche and ability to also grow in pure culture on wheat straw, protocols for transformation and antibiotic selection of the strain were established. Hygromycin was found to be a reliable selectable transformation marker, and the mammalian codon-optimized green fluorescent protein was expressed and used to visualize fluorescence in transformed cells of strain 2T2.1.

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