0031 - Genetic manipulation of marine bacteria: set up of a method for insertional mutagenesis in Halomonas aquamarina 9B

2018 ◽  
Author(s):  
Sara Borin ◽  
Elena Crotti ◽  
Francesca Mapelli
Keyword(s):  
Marine Bacteria ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Set Up

scholarly journals Analysis of the light intensity dependence of the growth ofSynechocystisand of the light distribution in a photobioreactor energized by 635 nm light

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5256 ◽  
Author(s):  
Alessandro Cordara ◽  
Angela Re ◽  
Cristina Pagliano ◽  
Pascal Van Alphen ◽  
Raffaele Pirone ◽  
...  
Keyword(s):  
Light Intensity ◽  
Photosynthetic Activity ◽  
Genetic Manipulation ◽  
Incident Light ◽  
Red Light ◽  
Clear Correlation ◽  
Heterogeneous Distribution ◽  
Cultivation System ◽  
Modelling Studies ◽  
Set Up

Synechocystisgathered momentum in modelling studies and biotechnological applications owing to multiple factors like fast growth, ability to fix carbon dioxide into valuable products, and the relative ease of genetic manipulation.Synechocystisphysiology and metabolism, and consequently, the productivity ofSynechocystis-based photobioreactors (PBRs), are heavily light modulated. Here, we set up a turbidostat-controlled lab-scale cultivation system in order to study the influence of varying orange–red light intensities onSynechocystisgrowth characteristics and photosynthetic activity.Synechocystisgrowth and photosynthetic activity were found to raise as supplied light intensity increased up to 500 μmol photons m−2s−1and to enter the photoinhibition state only at 800 μmol photons m−2s−1. Interestingly, reverting the light to a non-photo-inhibiting intensity unveiledSynechocystisto be able to promptly recover. Furthermore, our characterization displayed a clear correlation between variations in growth rate and cell size, extending a phenomenon previously observed in other cyanobacteria. Further, we applied a modelling approach to simulate the effects produced by varying the incident light intensity on its local distribution within the PBR vessel. Our model simulations suggested that the photosynthetic activity ofSynechocystiscould be enhanced by finely regulating the intensity of the light incident on the PBR in order to prevent cells from experiencing light-induced stress and induce their exploitation of areas of different local light intensity formed in the vessel. In the latter case, the heterogeneous distribution of the local light intensity would allowSynechocystisfor an optimized usage of light.

scholarly journals Assessing Stealth and Sensed Base Editing in Human Hematopoietic Stem/Progenitor Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3976-3976
Author(s):  
Martina Fiumara ◽  
Samuele Ferrari ◽  
Attya Omer ◽  
Stefano Beretta ◽  
Luisa Albano ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Single Strand ◽  
Clinical Translation ◽  
Hematopoietic Stem ◽  
Single Strand Break ◽  
Base Editing ◽  
Cas9 Nuclease

Abstract Genome editing represents a promising tool to manipulate human hematopoietic stem and progenitor cells (HSPCs) opening the possibility to correct hematopoietic diseases avoiding the risk of insertional mutagenesis and uncontrolled expression of the transgene, issues that emerged with retroviral and lentiviral gene therapy. Engineered nucleases such as CRISPR/Cas9 have enable targeted genetic manipulation in human HSPCs for therapeutic purposes. Still, nuclease-induced DNA double-strand breaks (DSBs) trigger p53-dependent DNA damage response affecting HSPC properties and may lead to unintended chromosomal rearrangements. Base editing (BE) holds the promise for precise editing by the introduction of specific single-nucleotide variants (SNVs) while bypassing the requirement for DSBs. In particular, base editors are composed by: i) a deamination domain that directly modifies nucleotides comprised within a defined editing window in one of the two genomic strands, and ii) a nickase Cas9 that introduces a single-strand break (SSB) on the other strand to promote more efficient base editing. Depending on the type of modification introduced editors are classified in Cytosine (C-) BE (C-G transition to T-A) and Adenine (A-) BE (A-T transition to G-C). However, a comprehensive characterization of efficiency, tolerability and genotoxicity of CBE and ABE in human HSPCs is lacking and is required to instruct the rationale towards safe and effective clinical translation. Here, we developed an optimized mRNA-based protocol for BE in human HSPCs and compared CBE4max, ABE8.20-m and Cas9 nuclease by targeting the same locus (B2M) using the same sgRNA. Common outcome for all editors is disruption of targeted gene expression, which is measured by flow cytometry and Next Generation Sequencing. ABE8.20-m showed higher efficiency than CBE4max and Cas9 nuclease at the target locus (up to 90, 40 and 50%), which was consistent across HSPC subpopulations comprising the most primitive compartment endowed with long term repopulation potential and cell sources (such as cord blood- and mobilized peripheral blood-derived HSPCs). Importantly, Cas9, but not CBE4max and ABE8.20m, treated HSPCs showed lower in-vitro clonogenic capacity than mock electroporated cells. Transcriptional analyses uncovered that CBE4max, but not ABE8.20-m, triggered p53 pathway activation, albeit at lower extent as compared to Cas9 and presumably consequent to a fraction of single-strand nicks turning into DSB upon DNA replication. Additionally, BE, and particularly CBE4max, upregulated the expression of interferon-stimulated genes, which was not ascribed to mRNA delivery. Remarkably, despite edited HSPCs showed long-term multilineage capacity in xenotransplanted mice, CBE4max edited cells tended to decrease over time in the graft pointing to some detrimental response to the treatment of the long-term engrafting HSC subset. Overall, our results prompt further investigation on BE sensing in human HSPCs. On-going studies are aimed to investigate clonal dynamics and genome integrity of base-edited HSPCs with the final goal of building confidence for their perspective clinical translation. Disclosures Naldini: Genenta Science: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

scholarly journals Methods for the genetic manipulation of marine bacteria

2018 ◽  
Vol 33 ◽  
pp. 17-28 ◽  
Author(s):  
Zahraa Zeaiter ◽  
Francesca Mapelli ◽  
Elena Crotti ◽  
Sara Borin
Keyword(s):  
Marine Bacteria ◽  
Genetic Manipulation

scholarly journals Multiple Degradation and Resistance Capabilities of Marine Bacteria Isolated from Niger Delta, Nigeria on Petroleum Pollutants and Heavy Metals

2019 ◽  
pp. 1-17
Author(s):  
Bright Obidinma Uba ◽  
Edna Ifeoma Chukwura ◽  
Ebere Linda Okoye ◽  
Onyedikachi Ubani ◽  
Mark Iyere Irabor ◽  
...  
Keyword(s):  
Heavy Metals ◽  
Marine Bacteria ◽  
Basal Medium ◽  
Cost Effective ◽  
Petroleum Products ◽  
Growth Effect ◽  
Bacterial Strains ◽  
Set Up ◽  
Rivers State ◽  
Petroleum Pollutants

Aims: To determine multiple degradation and resistance capabilities of marine bacteria isolated from Rivers State, Nigeria on petroleum pollutants and heavy metals. Study Design: Nine treatments and the controls designs were set up in triplicates containing              100 mL of sterile modified mineral basal medium in 250 mL conical flasks supplemented with 50, 100, 200 and 300 ppm of xylene, anthracene and pyrene each; 1 % of other petroleum pollutants and 300 ppm of heavy metals, nine marine hydrocarbon degraders and incubated at 24ºC for 5 - 7 days. The nine treatments and controls set ups designated as ANT1, XYL2, PYR3, ANT4, PYR5, ANT6, XYL7, XYL8, PYR9 and CTRL (Without hydrocarbons) were used to determine the multiple degradability of the marine bacteria.   Place and Duration of Study: Department of Microbiology, Faculty of Natural Sciences, Chukwuemeka Odumegwu Ojukwu University, Uli Nigeria between September, 2014 and March, 2017. Methodology: A laboratory scale study was carried on six composite samples of the sediment and water samples from the three studied areas using enrichment, screening, selection, molecular, growth effect and substrate specificity techniques. Results: The findings revealed that screening and selection for the indigenous bacterial isolates from the three studied areas resulted in the isolation of nine out of forty eight (9/48) of the potent strains representing 18.75 % of the total isolates with significant (P = .05) multiple degradation and resistance potentials but with different efficiencies on xylene, anthracene and pyrene, other petroleum products and heavy metals at 50 – 300 pm and 1 %. All the nine potent strains were fully characterized molecularly and phylogenetically and belong to the genera: Providencia, Alcaligenes, Brevundimonas, Myroides, Serratia, and Bacillus. Conclusion: Thus, these selected potent bacterial strains could significantly contribute in the development of a cost - effective bioremediation process on aromatic hydrocarbons and heavy metals contaminated environments in Nigeria.

scholarly journals An exploration of the rapid transformation method for Dunaliella salina system

AMB Express ◽  
2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Guannan Song ◽  
Wan Wang ◽  
Lina Hu ◽  
Yu Liu ◽  
Aifang Li ◽  
...  
Keyword(s):  
Dunaliella Salina ◽  
Genetic Manipulation ◽  
Expression System ◽  
Transformation Method ◽  
Pcr Analysis ◽  
Foreign Gene ◽  
High Efficient ◽  
Set Up ◽  
Transformation Methods ◽  
Rapid Transformation

Abstract As a new expression system, Dunaliella salina (D. salina) has bright prospects and applications in various fields. However, its application is currently restricted because of the low expression and instability of foreign gene in D. salina cells. During genetic operation, transformation is a crucial step for genes expression in D. salina system. Although several transformation methods are existing currently, many inherent deficiencies and limitations still can be found in actual practice. Thus, we attempted to set up a rapid transformation method using the change of salt concentrations for D. salina. Based on osmotic pressure difference, exogenous genes can be spontaneously transferred into D. salina cells. After that, transformed D. salina cells were subjected to histochemical and molecular analysis. The results showed that the reporter gene, beta-glucuronidase genes were successfully expressed in the positive transformants, and detected in all of transformed cells by PCR analysis. Moreover, different transformation parameters, containing the salt gradient, time, dye dosage and Triton X-100 concentration, were optimized to obtain an optimal transformation result. Taken together, we preliminarily established a rapid transformation method with the features of fast, simple, economic, and high-efficient. This method will provide a strong genetic manipulation tool for the future transformation of D. salina system.

scholarly journals Development of Safer Gene Delivery Systems to Minimize the Risk of Insertional Mutagenesis-Related Malignancies: A Critical Issue for the Field of Gene Therapy

ISRN Oncology ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Gaetano Romano
Keyword(s):  
Gene Delivery ◽  
Mammalian Cells ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Shuttle Vector ◽  
Severe Combined Immunodeficiency ◽  
Sleeping Beauty ◽  
Delivery Systems ◽  
Vector System ◽  
Chromosomal Dna

Integrating gene delivery systems allow for a more stable transgene expression in mammalian cells than the episomal ones. However, the integration of the shuttle vector within the cellular chromosomal DNA is associated with the risk of insertional mutagenesis, which, in turn, may cause malignant cell transformation. The use of a retroviral-derived vector system was responsible for the development of leukemia in five children, who participated in various clinical trials for the treatment of severe combined immunodeficiency (SCID-X1) in France and in the United Kingdom. Unfortunately, the hematological malignancy claimed the life of one patient in 2004, who was enrolled in the French clinical trial. In addition, adeno-associated-viral-(AAV-) mediated gene transfer induced tumors in animal models, whereas the Sleeping Beauty (SB) DNA transposon system was associated with insertional mutagenesis events in cell culture systems. On these grounds, it is necessary to develop safer gene delivery systems for the genetic manipulation of mammalian cells. This paper discusses the latest achievements that have been reported in the field of vector design.

scholarly journals Broad Purpose Vector for Site-Directed Insertional Mutagenesis in Bifidobacterium breve

2021 ◽  
Vol 12 ◽  
Author(s):  
Emily C. Hoedt ◽  
Francesca Bottacini ◽  
Nora Cash ◽  
Roger S. Bongers ◽  
Kees van Limpt ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Base Substitution ◽  
Gene Encoding ◽  
Bifidobacterium Breve ◽  
Suicide Vector ◽  
Dehydrogenase Gene ◽  
Restriction Sites ◽  
Target Strain ◽  
Restriction Modification

Members of the genus Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an α-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.

scholarly journals Agrobacterium-Mediated Transformation of Fusarium oxysporum: An Efficient Tool for Insertional Mutagenesis and Gene Transfer

2001 ◽  
Vol 91 (2) ◽  
pp. 173-180 ◽  
Author(s):  
E. D. Mullins ◽  
X. Chen ◽  
P. Romaine ◽  
R. Raina ◽  
D. M. Geiser ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Fusarium Oxysporum ◽  
Fungal Pathogens ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Fungal Spores ◽  
Hygromycin B ◽  
Fungal Transformation ◽  
Efficient Tool ◽  
Binary Vectors

Agrobacterium tumefaciens-mediated transformation (ATMT) has long been used to transfer genes to a wide variety of plants and has also served as an efficient tool for insertional mutagenesis. In this paper, we report the construction of four novel binary vectors for fungal transformation and the optimization of an ATMT protocol for insertional mutagenesis, which permits an efficient genetic manipulation of Fusarium oxysporum and other phytopathogenic fungi to be achieved. Employing the binary vectors, carrying the bacterial hygromycin B phosphotrans-ferase gene (hph) under the control of the Aspergillus nidulans trpC promoter as a selectable marker, led to the production of 300 to 500 hygromycin B resistant transformants per 1 × 106 conidia of F. oxysporum, which is at least an order of magnitude higher than that previously accomplished. Transformation efficiency correlated strongly with the duration of cocultivation of fungal spores with Agrobacterium tumefaciens cells and significantly with the number of Agrobacteruium tumefaciens cells present during the cocultivation period (r = 0.996; n = 3; P < 0.01). All transformants tested remained mitotically stable, maintaining their hygromycin B resistance. Growing Agrobacterium tumefaciens cells in the presence of acetosyringone (AS) prior to cocultivation shortened the time required for the formation of transformants but decreased to 53% the percentage of transformants containing a single T-DNA insert per genome. This increased to over 80% when Agrobacterium tumefaciens cells grown in the absence of AS were used. There was no correlation between the average copy number of T-DNA per genome and the colony diameter of the transformants, the period of cocultivation or the quantity of Agrobacterium tumefaciens cells present during cocultivation. To isolate the host sequences flanking the inserted T-DNA, we employed a modified thermal asymmetric interlaced PCR (TAIL-PCR) technique. Utilizing just one arbitrary primer resulted in the successful amplification of desired products in 90% of those transformants analyzed. The insertion event appeared to be a random process with truncation of the inserted T-DNA, ranging from 1 to 14 bp in size, occurring on both the right and left border sequences. Considering the size and design of the vectors described here, coupled with the efficiency and flexibility of this ATMT protocol, it is suggested that ATMT should be regarded as a highly efficient alternative to other DNA transfer procedures in characterizing those genes important for the pathogenicity of F. oxysporum and potentially those of other fungal pathogens.

Transgenesis of schistosomes: approaches employing mobile genetic elements

Parasitology ◽  
2007 ◽  
Vol 135 (2) ◽  
pp. 141-153 ◽  
Author(s):  
V. H. MANN ◽  
M. E. MORALES ◽  
K. J. KINES ◽  
P. J. BRINDLEY
Keyword(s):  
Large Scale ◽  
Insertional Mutagenesis ◽  
Genetic Manipulation ◽  
Draft Genome ◽  
Mobile Genetic Elements ◽  
Forward Genetics ◽  
Loss Of Function ◽  
Genetic Elements ◽  
The Past ◽  
Foreign Genes

SUMMARYDraft genome sequences forSchistosoma mansoniandSchistosoma japonicumare now available. However, the identity and importance of most schistosome genes have yet to be determined. Recently, progress has been made towards the genetic manipulation and transgenesis of schistosomes. Both loss-of-function and gain-of-function approaches appear to be feasible in schistosomes based on findings described in the past 5 years. This review focuses on reports of schistosome transgenesis, specifically those dealing with the transformation of schistosomes with exogenous mobile genetic elements and/or their endogenous relatives for the genetic manipulation of schistosomes. Transgenesis mediated by mobile genetic elements offers a potentially tractable route to introduce foreign genes to schistosomes, a means to determine the importance of schistosome genes, including those that could be targeted in novel interventions and the potential to undertake large-scale forward genetics by insertional mutagenesis.

The Effects of Drying Techniques on Clay-Rich Soil Texture

1974 ◽  
Vol 32 ◽  
pp. 466-467
Author(s):  
T. G. Naymik
Keyword(s):  
Critical Point ◽  
Liquid Nitrogen ◽  
Liquid Nitrogen Temperature ◽  
Drying Methods ◽  
Air Drying ◽  
Freeze Dried ◽  
Critical Point Drying ◽  
Set Up ◽  
The Relationship ◽  
Quartz Grains

Three techniques were incorporated for drying clay-rich specimens: air-drying, freeze-drying and critical point drying. In air-drying, the specimens were set out for several days to dry or were placed in an oven (80°F) for several hours. The freeze-dried specimens were frozen by immersion in liquid nitrogen or in isopentane at near liquid nitrogen temperature and then were immediately placed in the freeze-dry vacuum chamber. The critical point specimens were molded in agar immediately after sampling. When the agar had set up the dehydration series, water-alcohol-amyl acetate-CO2 was carried out. The objectives were to compare the fabric plasmas (clays and precipitates), fabricskeletons (quartz grains) and the relationship between them for each drying technique. The three drying methods are not only applicable to the study of treated soils, but can be incorporated into all SEM clay soil studies.

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