Protocol: Precision engineering of plant gene loci by homologous recombination cloning in Escherichia coli.

© 2005 Roden et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Plant genome sequence data now provide opportunities to conduct molecular genetic studies at the level of the whole gene locus and above. Such studies will be greatly facilitated by adopting and developing further the new generation of genetic engineering tools, based on homologous recombination cloning in Escherichia coli, which are free from the constraints imposed by the availability of suitably positioned restriction sites. Here we describe the basis for homologous recombination cloning in E. coli, the available tools and resources, together with a protocol for long range cloning and manipulation of an Arabidopsis thaliana gene locus, to create constructs coordinately driven by locus-specific regulatory elements.Peer reviewe

Molecular Cloning of Gonadotropin Hormones I (Gth-i) and II (Gth-ii) Genes in the Hard-lipped Barb (Osteochillus Hasseltii) and the Effect of Photoperiods on the Genes Expression

Photoperiods is one of the factors that influence regulation on endocrine gland in producing hormones necessary for gonadal growth and development, gametogenesis and reproductive cycles in fish reproduction. This study clones the gonadotropin hormones I (GtH-I) and II (GtH-II) genes in hard-lipped bard, and investigates the effect of photoperiod on the genes expression. Experiment was designed using three treatments; 14L: 10D (control), 8L: 16D (short photoperiods), and 18L: 6D (long photoperiods). Four aquariums with nine fish/tank were used, serving as replicates. Fish were kept under this photoperiods treatment for eight weeks. Pituitary activities were observed by GtH genes expression measurement. The length of cDNA GtH-I was 222 bp, and the cDNA GtH-II was 354 bp. The GtH-I precursors encoded by cDNA consisted of 67 amino acids, including mature peptide. The level of GtH gene expression was significantly increased as longer photoperiods given (P<0.05). The results indicated that hard-lipped barb reproductive performance is affected by photoperiods treatment

Molecular Cloning of Sucrose Isomerase Gene and Agrobacterium-Mediated Genetic Transformation of Potato (Solanum tuberosum L.) Plants

Potato (Solanum tuberosum L.) is one of the most common and important food sources on the planet, and they essential as a staple dietary item for much of the world's population. Potatoes contain carbohydrates, which lead to high blood sugar. Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a functional isomer of sucrose its non-cariogenicity low calorific value and it is an ideal sugar substitute to use in food production. The sucrose isomerase (palI) gene that is obtained from Erwinia rhapontici is one of the most common genes that can convert sucrose into palatinose. In present study, pQE-30- palI construct was succeffuly transformed and expression into E. coli. Sucrose isomerase (palI) gene was cloned and overexpressed into a plant expression vector pBinAR- palI contains sucrose isomerase gene (palI) fused to proteinase inhibitor II signal sequence under CaMV-35S promoter and Octopine Synthase (OCS) terminator. Expression of the protein was verified by western blot assay. Also, expression of the palI gene within the apoplast of transgenic tubers under control of a tuber-specific patatin class I B33 promoter instigated quantitative conversion of sucrose into palatinose. Tuber extracts from potato cv. Désirée were analyzed for their soluble carbohydrate composition using HPLC

QuickStep-Cloning: a sequence-independent, ligation-free method for rapid construction of recombinant plasmids

Background Molecular cloning is an essential step in biological engineering. Methods involving megaprimer-based PCR of a whole plasmid are promising alternatives to the traditional restriction-ligation-based molecular cloning. Their widespread use, however, is hampered by some of their inherent characteristics, e.g., linear amplification, use of self-annealing megaprimers and difficulty with performing point insertion of DNA. These limitations result in low product yield and reduced flexibility in the design of a genetic construct. Result Here, we present a novel technique of directional cloning, which overcomes these problems yet retaining the simplicity of whole-plasmid amplification. QuickStep-Cloning utilizes asymmetric PCRs to create a megaprimer pair with 3′-overhangs, and hence, facilitates the subsequent exponential whole-plasmid amplification. QuickStep-Cloning generates nicked-circular plasmids, thereby permitting direct bacterial transformation without DNA ligation. It allows DNA fragment integration into any plasmid at any position, in an efficient, time- and cost-effective manner, without tedious intermediate DNA gel purification, modified oligonucleotides, specialty enzymes and ultra-competent cells. The method is compatible with competent E. coli cells prepared using the conventional calcium chloride method. Conclusion QuickStep-Cloning expands the versatility of megaprimer-based cloning. It is an excellent addition to the cloning toolbox, for the benefit of protein engineers, metabolic engineers and synthetic biologists

Engineering a minimal cloning vector from a pUC18 plasmid backbone with an extended multiple cloning site

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described

Diversity of actinobacteria in the marshes of Ezzemoul and Djendli in northeastern Algeria

The main purpose of this research is to study the microbial diversity of actinobacteria, living in “Ezzemoul” and “Djendli” sebkhas soils. These salt lakes are situated in the east of Algeria and they are microbiologically underexploited. Such unexplored ecological niches have been considered by many authors as sources of novel actinobacteria and bioactive molecules. Actinobacteria play an important role in safeguarding the environment by improving plant growth through nitrogen fixation, biodegradation, and bioremediation. Therefore, studying the diversity and distribution of actinobacteria in such special environments is important for determining the ecological and biotechnological roles of these microorganisms. In this article, we focused on the occurrence and the diversity of actinobacteria from sebkhas using two techniques: cultural and culture-independent (molecular cloning). The latter are based on phylogenetic analysis of the 16S rDNA gene. Thus, the cultural method allowed us to obtain 62 isolates: 40 from the “Ezzemoul” site and 22 from the “Djendli” site. These isolates tolerate mainly 2, 5, and 10% sodium chloride (NaCl) and belong to the genera Nocardiopsis, Streptomyces, and Rhodococcus. Moreover, the molecular cloning gave us 39 clones. Twenty-four clone sequences from “Ezzemoul” site are affiliated to the genera Demequina, Plantactinospora, Friedmanniella, and Mycobacterium. Also, 15 clone sequences from “Djendli” site are related to the genera Marmoricola, Phytoactinopolyspora, Streptomyces, and to an unclassified actinobacterial clone. Some sequences from both sites are related to uncultured clones. In addition to the data provided by the cultural method, molecular cloning allowed us to have additional information about the unknown actinobacteria, uncultured ones as well as on the genera that exist in both sites. So, the cultural method is complementary to the culture-independent one, and their combination revealed an important diversity in targeted saline environments. Furthermore, all new isolated strains that tolerate 10% NaCl may have a very interesting biotechnological potential in the future

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation

Ion channels: structural basis for function and disease.

Ion channels are ubiquitous proteins that mediate nervous and muscular function, rapid transmembrane signaling events, and ionic and fluid balance. The cloning of genes encoding ion channels has led to major strides in understanding the mechanistic basis for their function. These advances have shed light on the role of ion channels in normal physiology, clarified the molecular basis for an expanding number of diseases, and offered new direction to the development of rational therapeutic interventions

CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method.

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate

Molecular cloning and characterization of B-cadherin, a novel chick cadherin.

Calcium-dependent cell-cell adhesion is mediated in large part by a set of homologous integral membrane glycoproteins termed cadherins. In this report, antibodies to conserved domains in previously described cadherins have been used to isolate cDNAs encoding a novel chick cadherin. The deduced primary structure of this novel molecule, assigned the name B-cadherin, contains 726 amino acid residues which include five extracellular domains characteristic of this class of adhesion molecules, a single putative transmembrane spanning region, and a cytoplasmic tail. In each domain, B-cadherin shares extensive homologies with other cadherins, but is more closely related to E-cadherin, P-cadherin, and L-CAM than to N-cadherin. It is expressed in a wide variety of chick tissues at embryonic day 13. In particular, immunohistochemical staining and in situ hybridization localize B-cadherin protein and mRNA to the epithelial lining of the choroid plexus and to cells in specific layers of the optic tectum in chick brain. Levels of the protein and RNA transcript change dramatically as development proceeds in chick brain. These results suggest that B-cadherin has important functions in neurogenesis, in at least some epithelia, and in embryogenesis