scholarly journals Saturation mutagenesis genome engineering of infective ΦX174 bacteriophage via unamplified oligo pools and golden gate assembly

2019 ◽  
Author(s):  
Matthew S. Faber ◽  
James T. Van Leuven ◽  
Martina M. Ederer ◽  
Yesol Sapozhnikov ◽  
Zoë L. Wilson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Genome Engineering ◽  
Reverse Genetics ◽  
Viral Evolution ◽  
Single Site ◽  
Saturation Mutagenesis ◽  
Golden Gate ◽  
Genes Encoding ◽  
Site Mutagenesis ◽  
Golden Gate Cloning

Here we present a novel protocol for the construction of saturation single-site—and massive multi-site—mutant libraries of a bacteriophage. We segmented the ΦX174 genome into 14 non-toxic and non-replicative fragments compatible with golden gate assembly. We next used nicking mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near-comprehensive single-site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11,860 programmed mutations. Golden Gate cloning was then used to assemble the complete ΦX174 mutant genome and generate libraries of infective viruses. This protocol will enable reverse genetics experiments for studying viral evolution and, with some modifications, can be applied for engineering of therapeutically relevant bacteriophages with larger genomes.

scholarly journals Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2542
Author(s):  
Won-Nyeong Kim ◽  
Hye-Jeong Kim ◽  
Young-Soo Chung ◽  
Hyun-Uk Kim
Keyword(s):  
Reverse Genetics ◽  
Restriction Enzymes ◽  
Golden Gate ◽  
Knock Out ◽  
Guide Rnas ◽  
Genetics Research ◽  
Multiple Genes ◽  
Cas9 Protein ◽  
Cloning Method ◽  
Golden Gate Cloning

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.

scholarly journals Golden Mutagenesis: An efficient multi-site-saturation mutagenesis approach by Golden Gate cloning with automated primer design

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Pascal Püllmann ◽  
Chris Ulpinnis ◽  
Sylvestre Marillonnet ◽  
Ramona Gruetzner ◽  
Steffen Neumann ◽  
...  
Keyword(s):  
Primer Design ◽  
Saturation Mutagenesis ◽  
Golden Gate ◽  
Site Saturation ◽  
Golden Gate Cloning

scholarly journals Saturation Mutagenesis Genome Engineering of Infective ΦX174 Bacteriophage via Unamplified Oligo Pools and Golden Gate Assembly

2019 ◽  
Vol 9 (1) ◽  
pp. 125-131 ◽  
Author(s):  
Matthew S. Faber ◽  
James T. Van Leuven ◽  
Martina M. Ederer ◽  
Yesol Sapozhnikov ◽  
Zoë L. Wilson ◽  
...  
Keyword(s):  
Genome Engineering ◽  
Saturation Mutagenesis ◽  
Golden Gate

scholarly journals Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Copy Number ◽  
Life Science ◽  
Insertion Site ◽  
Golden Gate ◽  
Common Elements ◽  
Modern Life ◽  
Specific Host ◽  
Modular Cloning ◽  
Golden Gate Cloning ◽  
Selection Of

Abstract Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

Opposite outcomes of the within-host competition between highly and low pathogenic H5N8 avian influenza viruses in chickens compared to ducks

2021 ◽  
Author(s):  
Pierre Bessière ◽  
Thomas Figueroa ◽  
Amelia Coggon ◽  
Charlotte Foret-Lucas ◽  
Alexandre Houffschmitt ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Reverse Genetics ◽  
Highly Pathogenic Avian Influenza ◽  
Viral Evolution ◽  
Influenza Viruses ◽  
Poultry Production ◽  
Avian Influenza Viruses ◽  
Highly Pathogenic ◽  
Pathogenic Avian Influenza ◽  
Low Pathogenic Avian Influenza

Highly pathogenic avian influenza viruses (HPAIV) emerge from low pathogenic avian influenza viruses (LPAIV) through the introduction of basic amino acids at the hemagglutinin (HA) cleavage site. Following viral evolution, the newly formed HPAIV likely represents a minority variant within the index host, predominantly infected with the LPAIV precursor. Using reverse-genetics engineered H5N8 viruses differing solely at the HA cleavage, we tested the hypothesis that the interaction between the minority HPAIV and the majority LPAIV could modulate the risk of HPAIV emergence and that the nature of the interaction could depend on the host species. In chickens, we observed that the H5N8 LP increased H5N8 HP replication and pathogenesis. By contrast, the H5N8 LP antagonized H5N8 HP replication and pathogenesis in ducks. Ducks mounted a more potent antiviral innate immune response than chickens against the H5N8 LP , which correlated with H5N8 HP inhibition. These data provide experimental evidence that HPAIV may be more likely to emerge in chickens than in ducks and underscore the importance of within-host viral variants interactions in viral evolution. IMPORTANCE Highly pathogenic avian influenza viruses represent a threat to poultry production systems and to human health because of their impact on food security and because of their zoonotic potential. It is therefore crucial to better understand how these viruses emerge. Using a within-host competition model between highly and low pathogenic avian influenza viruses, we provide evidence that highly pathogenic avian influenza viruses could be more likely to emerge in chickens than in ducks. These results have important implications for highly pathogenic avian influenza virus emergence prevention and they underscore the importance of within-host viral variants interactions in virus evolution.

scholarly journals Cloning recombinant pHW2000 vector carrying the HA gene for preparation of the primary influenza A/H5N1 vaccine strain

2017 ◽  
Vol 33 (1S) ◽  
Author(s):  
Nguyen Thi Thu Hang ◽  
Hoang Thi Thu Hang ◽  
Nguyen Hung Chi ◽  
Chu Hoang Ha ◽  
Nguyen Trung Nam
Keyword(s):  
Amino Acids ◽  
Avian Influenza ◽  
Influenza A ◽  
Reverse Genetics ◽  
Genetic Relationships ◽  
Highly Pathogenic ◽  
Ha Gene ◽  
H5n1 Influenza ◽  
Hpai H5n1 ◽  
Clade 2.3.2.1C

Influenza A/H5N1 virus evolves rapidly and generate new variants, therefore it is essential to develop effective vaccines against the currently circulating influenza strains. Among clades and subclades of highly pathogenic avian influenza (HPAI) H5N1 viruses circulating in Vietnam, H5N1 clade 1.1 and clade 2.3.2.1c possess genetic relationships to many strains of influenza; thus they are suggested to be used for producing vaccines against avian influenza. In this article, two HA gene segments of two types of A/H5N1 influenza clade have been designed: HA clade 1.1 gene consists 1825 nucleotides encoding 565 amino acids, HA clade 2.3.2.1c gene consists 1822 nucleotides, encoding 564 amino acids. Most importantly, nucleotide sequence of the pathogenic region of HA was removed. Each of the two HA segments corresponding to the two clades were successfully cloned into pHW2000 vector and will be used as a candidate for production of avian influenza vaccines using reverse genetics technique.  

Consistency and Change

2019 ◽  
Author(s):  
Alan Kelly
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Living Organism ◽  
Peptide Bond ◽  
The Body ◽  
Blood Proteins ◽  
Genes Encoding ◽  
Fundamental Phenomenon ◽  
P Proteins

Proteins are, in my view, the most impressive molecules in food. They influence the texture, crunch, chew, flow, color, flavor, and nutritional quality of food. Not only that, but they can radically change their properties and how they behave depending on the environment and, critically for food, in response to processes like heating. Even when broken down into smaller components they are important, for example giving cheese many of its critical flavor notes. Indeed, I would argue that perhaps the most fundamental phenomenon we encounter in cooking or processing food is the denaturation of proteins, as will be explained shortly. Beyond food, the value of proteins and their properties is widespread across biology. Many of the most significant molecules in our body and that of any living organism (including plants and animals) are proteins. These include those that make hair and skin what they are, as well as the hemoglobin that transports oxygen around the body in our blood. Proteins are built from amino acids, a family of 20 closely related small molecules, which all have in chemical terms the same two ends (chemically speaking, an amino end and an acidic end, hence the name) but differ in the middle. This bit in the middle varies from amino acid to amino acid, from simple (a hydrogen atom in the case of glycine, the simplest amino acid) to much more complex structures. Amino acids can link up very neatly, as the amino end of one can form a bond (called a peptide bond) with the acid end of another, and so forth, so that chains of amino acids are formed that, when big enough (more than a few dozen amino acids), we call proteins. Our bodies produce thousands of proteins for different functions, and the instructions for which amino acids combine to make which proteins are essentially what the genetic code encrypted in our DNA specifies. We hear a lot about our genes encoding the secrets of life, but what that code spells is basically P-R-O-T-E-I-N. Yes, these are very important molecules!

scholarly journals Transcriptomic Analysis of Staphylococcus xylosus in Solid Dairy Matrix Reveals an Aerobic Lifestyle Adapted to Rind

2020 ◽  
Vol 8 (11) ◽  
pp. 1807
Author(s):  
Sabine Leroy ◽  
Sergine Even ◽  
Pierre Micheau ◽  
Anne de La Foye ◽  
Valérie Laroute ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Antioxidant Enzymes ◽  
Dairy Products ◽  
Molecular Mechanisms ◽  
Iron Acquisition ◽  
Carbon Sources ◽  
Original System ◽  
Staphylococcus Xylosus ◽  
Genes Encoding

Staphylococcus xylosus is found in the microbiota of traditional cheeses, particularly in the rind of soft smeared cheeses. Despite its frequency, the molecular mechanisms allowing the growth and adaptation of S. xylosus in dairy products are still poorly understood. A transcriptomic approach was used to determine how the gene expression profile is modified during the fermentation step in a solid dairy matrix. S. xylosus developed an aerobic metabolism perfectly suited to the cheese rind. It overexpressed genes involved in the aerobic catabolism of two carbon sources in the dairy matrix, lactose and citrate. Interestingly, S. xylosus must cope with nutritional shortage such as amino acids, peptides, and nucleotides, consequently, an extensive up-regulation of genes involved in their biosynthesis was observed. As expected, the gene sigB was overexpressed in relation with general stress and entry into the stationary phase and several genes under its regulation, such as those involved in transport of anions, cations and in pigmentation were up-regulated. Up-regulation of genes encoding antioxidant enzymes and glycine betaine transport and synthesis systems showed that S. xylosus has to cope with oxidative and osmotic stresses. S. xylosus expressed an original system potentially involved in iron acquisition from lactoferrin.

scholarly journals Human Cancer-Associated Mutations of SF3B1 Lead to a Splicing Modification of Its Own RNA

Cancers ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 652
Author(s):  
Tiffany Bergot ◽  
Eric Lippert ◽  
Nathalie Douet-Guilbert ◽  
Séverine Commet ◽  
Laurent Corcos ◽  
...  
Keyword(s):  
Amino Acids ◽  
Human Cancer ◽  
Myeloid Cell ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Small Nuclear Ribonucleoprotein ◽  
Genes Encoding ◽  
Protein Isoform ◽  
Nuclear Ribonucleoprotein

Deregulation of pre-mRNA splicing is observed in many cancers and hematological malignancies. Genes encoding splicing factors are frequently mutated in myelodysplastic syndromes, in which SF3B1 mutations are the most frequent. SF3B1 is an essential component of the U2 small nuclear ribonucleoprotein particle that interacts with branch point sequences close to the 3’ splice site during pre-mRNA splicing. SF3B1 mutations mostly lead to substitutions at restricted sites in the highly conserved HEAT domain, causing a modification of its function. We found that SF3B1 was aberrantly spliced in various neoplasms carrying an SF3B1 mutation, by exploring publicly available RNA sequencing raw data. We aimed to characterize this novel SF3B1 transcript, which is expected to encode a protein with an insertion of eight amino acids in the H3 repeat of the HEAT domain. We investigated the splicing proficiency of this SF3B1 protein isoform, in association with the most frequent mutation (K700E), through functional complementation assays in two myeloid cell lines stably expressing distinct SF3B1 variants. The yeast Schizosaccharomyces pombe was also used as an alternative model. Insertion of these eight amino acids in wild-type or mutant SF3B1 (K700E) abolished SF3B1 essential function, highlighting the crucial role of the H3 repeat in the splicing function of SF3B1.

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