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Vector Technology of Relevance to Nitrogen Fixation Research*
Published in Peter M. Gresshoff, Molecular Biology of Symbiotic Nitrogen Fixation, 2018
Reinhard Simon, Ursula B. Priefer
Some of the general-purpose cloning vectors described in the previous section may be used in this context. For example, since the EcoRI and SstI cloning sites of pKT230 are within the Sm gene, inserts into these sites can be expressed under the control of the Sm promoter. Hence, by cloning a given gene, one can have either constitutive expression from the vector promoter or expression from a promoter within the cloned fragment, depending on the orientation. This strategy has been applied in studies of the symbiotic genes of R. leguminosarum, e.g., for constitutive expression of the nodD gene.208
Methods in Molecular Biology
Published in Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman, Molecular Imaging in Oncology, 2008
In 1972, Cohen et al. developed a recombinant DNA technology that allowed DNA from one organism to be cloned into a carrier DNA molecule and be replicated and expressed in a new host (3). This technique, called molecular cloning, has revolutionized the field of molecular biology. DNA molecule used to carry a foreign DNA fragment into a bacterial or eukaryotic host organism is called cloning vector. There are several different types of vectors. The simplest and most commonly used DNA vector is derived from viral chromosomes and called plasmid. Plasmids are extrachromosomal, doubled-stranded circular molecules of DNA present in microorganisms, especially bacteria. They range from about 1 kilobase (kb) to over 200 kb in size, with an average 15 kb, and replicate autonomously. Other types of cloning vectors include cosmids, bacteriophages, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs).
Novel technologies to characterize and engineer the microbiome in inflammatory bowel disease
Published in Gut Microbes, 2022
Alba Boix-Amorós, Hilary Monaco, Elisa Sambataro, Jose C. Clemente
Classic tools to genetically engineer bacteria use recombinant plasmids as cloning vectors to deliver genes of interest. Cloning vectors allow the production of large amounts of protein, but embody certain limitations including low efficiency of transformation and limitations of insert size. Recent discoveries and development of new tools, such as the CRISPR-Cas systems, have revolutionized the genetic engineering scene, offering an enormous potential to engineer genomes with greater efficacy than previously achieved.223 CRISPR (clustered regularly interspaced short palindromic repeats) and its associated Cas proteins are tools derived from the prokaryotic immune system that have been co-opted as genetic editing tools.223,224 This methodology has been applied to bacteria and yeasts to modify their functional repertoire, exploited for industrial applications or used for the direct removal of specific genes or pathogens.222–225 Among the different CRISPR systems, CRISPR-Cas9 and CRISPR-Cas12a (also known as Cpf1) are two major nucleases that have been used in bacterial genetic editing experiments. By inserting a guide RNA sequence targeting a specific region of the bacterial DNA, Cas nucleases introduce a break in the pathogen’s genome which allows the removal of specific genes or causes bacterial death.223,226,227 If a template DNA is provided, the genomic break introduced by the Cas nucleases can be repaired by homologous recombination, inserting the new DNA fragment into the bacterial genome.
AhR/IL-22 pathway as new target for the treatment of post-infectious irritable bowel syndrome symptoms
Published in Gut Microbes, 2022
Maëva Meynier, Elodie Baudu, Nathalie Rolhion, Manon Defaye, Marjolène Straube, Valentine Daugey, Morgane Modoux, Ivan Wawrzyniak, Frédéric Delbac, Romain Villéger, Mathieu Méleine, Esther Borras Nogues, Catherine Godfraind, Nicolas Barnich, Denis Ardid, Philippe Poirier, Harry Sokol, Jean-Marc Chatel, Philippe Langella, Valérie Livrelli, Mathilde Bonnet, Frédéric Antonio Carvalho
In this study, a genetically modified lactic acid bacteria was used to locally deliver IL-22 cDNA expression vector directly in the intestine. This method allows an IL-22 low availability in intestinal epithelial cells. Lactococcus lactis is used as a lactic acid bacteria model because (i) its genome has been completely sequenced, (ii) it is easy to manipulate genetically, and (iii) several cloning vectors have been already developed.4 A study targeting the plasmid transfer efficiency using this L. lactis strain clearly showed that it is directly linked to the plasmid copy number level.56,57 Using this approach, we showed a local protective effect of IL-22 on mucosal epithelial barrier. Citrobacter rodentium infection disrupts intestinal permeability.58 The L. lactisIL-22 treatment restored colonic occludin mRNA level, which was decreased by infection, confirming the critical role of IL22 in maintaining intestinal barrier function.59 Such treatment improved CHS and anxiety-like behavior after pathogen clearance independently of infection-induced inflammation. Effects of infection and treatments were assessed with an original video-tracking device allowing a continuous and long-term monitoring of animal behavior, in a home cage-like setting. C. rodentium post-infected mice spent more time in the shelter zone during the dark period, which is positively associated with fear-related behavior and then be considered as anxiety- and depression-like troubles.60 Thus, the L. lactisIL-22 treatment favors healing of the main features caused by a C. rodentium infection in mice. These data confirmed the strong impact of AhR/IL-22 pathway deregulation in physiopathology of PI-IBS.
Yeast-inspired drug delivery: biotechnology meets bioengineering and synthetic biology
Published in Expert Opinion on Drug Delivery, 2019
Chinnu Sabu, Panakkal Mufeedha, Kannissery Pramod
Few types of yeast cloning vectors are seen, and they are so-called shuttle vectors. Shuttle vectors have the ability to replicate and be selected in both bacteria and yeast. Since plasmid preparation from yeast is ineffective, shuttle vectors are developed. Yeast cloning vectors based on 2-μm plasmid are called yeast episomal plasmids (YEPs). YEPs have the capability to replicate independently or can be integrated into one of the yeast chromosomes. They are considered as high copy number vectors consisting of a selective marker and origin of replication. The major disadvantages associated with their recombinants are they are highly unstable making it difficult and time-consuming to achieve a reliable result. Another type of yeast vectors is yeast integrative plasmids (YIPs). YIPs contain a selective marker and lack origin of replication. They cannot replicate independently and have a low transformational frequency. On the other hand, the recombinants are highly stable making it more beneficial. Yeast replicative plasmids are another type of cloning vectors. Their backbone consists of origin of replication in close proximity to the selective marker. They replicate independently with high transformational frequency. Like YEPs, their recombinants are highly unstable. As time passes, there was a large demand for large pieces of DNA to be manipulated. Yeast artificial chromosome (YAC) was developed at this time to combat the problem. YAC composes three main components; centromeres, the origin of replication and telomeres [43]. Researchers are still continuing to engineer yeast to produce eukaryotic proteins. The expressed foreign proteins can be used to synthesize life-saving drugs for the pharmaceutical industry. Yeast offers the simplicity of microbial growth and ability to perform post-translational modification [47]. In addition, they possess certain demerits such as expression of the heterologous protein and inability to perform certain post-translational modification [48].