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Studies on transformation of Escherichia coli with plasmids

Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.

https://www.pnas.org/content/pnas/82/2/488.full.pdf

Rapid and efficient site-specific mutagenesis without phenotypic selection.

Rapid and efficient site-specific mutagenesis without phenotypic selection

High efficiency transformation of Escherichia coli with plasmids

Plants can no longer be considered as standalone entities and a more holistic perception is needed. Indeed, plants harbor a wide diversity of microorganisms both inside and outside their tissues, in the endosphere and ectosphere, respectively. These microorganisms, which mostly
belong to Bacteria and Fungi, are involved in major functions such as plant nutrition and plant resistance to biotic and abiotic stresses. Hence, the microbiota impact plant growth and survival, two key components of fitness. Plant fitness is therefore a consequence of the plant per se and its microbiota, which collectively form a holobiont. Complementary to the reductionist perception of evolutionary pressures acting on plant or symbiotic compartments, the plant holobiont concept requires a novel perception of evolution. The interlinkages between the plant holobiont
components are explored here in the light of current ecological and evolutionary theories. Microbiome complexity and the rules of microbiotic community assemblage are not yet fully understood. It is suggested that the plant can modulate its microbiota to dynamically adjust to its
environment. To better understand the level of plant dependence on the microbiotic components, the core microbiota need to be determined at different hierarchical scales of ecology while pan-microbiome analyses would improve characterization of the functions displayed.

https://nph.onlinelibrary.wiley.com/doi/epdf/10.1111/nph.13312

The importance of the microbiome of the plant holobiont

DNA transfection of Escherichia coli by electroporation

Genome structure of the Lactobacillus temperate phage Φg1e: the whole genome sequence and the putative promoter/repressor system

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.

Akira Taketo

Papers

DNA transfection of Escherichia coli by electroporation

Genome structure of the Lactobacillus temperate phage Φg1e: the whole genome sequence and the putative promoter/repressor system

Cooperative Degradation of Chitin by Extracellular and Cell Surface-Expressed Chitinases from Paenibacillus sp. Strain FPU-7

Biochemical and Genetic Properties of PaenibacillusGlycosyl Hydrolase Having Chitosanase Activity and Discoidin Domain

Sensitivity of Escherichia coli to viral nucleic acid. V. Competence of calcium-treated cells.