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Results for “directed evolution”

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At least 1,801 records · Page 100Linked to original sources
PubMed2002

A tRNA aminoacylation system for non-natural amino acids based on a programmable ribozyme.

The ability to recognize tRNA identities is essential to the function of the genetic coding system. In translation aminoacyl-tRNA synthetases (ARSs) recognize the identities of tRNAs and charge them with their cognate amino acids. We show that an in vitro evolved ribozyme can also discriminate between specific tRNAs, and can transfer amino acids to the 3' ends of cognate tRNAs. The ribozyme interacts with both the CCA-3' terminus and the anticodon loop of tRNA(fMet), and its tRNA specificity is controlled by these interactions. This feature allows us to program the selectivity of the ribozyme toward specific tRNAs, and therefore to tailor effective aminoacyl-transfer catalysts. This method potentially provides a means of generating aminoacyl tRNAs that are charged with non-natural amino acids, which could be incorporated into proteins through cell-free translation.

Amino Acids↗
PubMed2002

Efficient gene targeting by homologous recombination in rice.

Modification of genes through homologous recombination, termed gene targeting, is the most direct method to characterize gene function. In higher plants, however, the method is far from a common practice. Here we describe an efficient and reproducible procedure with a strong positive/negative selection for gene targeting in rice, which feeds more than half of the world's population and is an important model plant. About 1% of selected calli and their regenerated fertile plants were heterozygous at the targeted locus, and only one copy of the selective marker used was found at the targeted site in their genomes. The procedure's applicability to other genes will make it feasible to obtain various gene-targeted lines of rice.

Base Sequence↗
PubMed2002

Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines.

We show that iterative antigen-mediated selection of B-cell lines that constitutively hypermutate their immunoglobulin V genes during culture can be exploited to generate antibodies in vitro. From Ramos, a hypermutating human B-cell line expressing IgM of unknown specificity, we derived descendants that exhibit stepwise improved binding to streptavidin. Binding is initially conferred by mutations in complementarity-determining regions (CDRs), but maturation is due to strategic framework mutations. A more powerful system is provided by a hypermutating chicken B-lymphoma line, owing to its rapid proliferation, high rate of mutation accumulation, and genetic tractability. Starting from a single cell, we selected parallel lineages of derivatives, making mutated antibodies of increasing affinity to independent test antigens. Selection is initiated at an exceedingly low affinity threshold, but antibodies can be delivered with nanomolar affinities. The strategy could prove useful for in vitro generation of antigen-specific monoclonal antibodies and may be extendable to the maturation of other protein-ligand interactions.

Animals↗
PubMed2005

Programming cellular function.

The process of cellular engineering is rapidly accelerating owing to advances in technologies to manipulate DNA and other biomolecules, giving rise to the field of synthetic biology. A meeting was held in August 2005 to present progress in the field and to discuss topics in ethics, safety and security.

Animals↗
PubMed2006

Training GFP to fold.

Explore the source record for details and available documents.

Directed Molecular Evolution↗
PubMed2006

SCALEs: multiscale analysis of library enrichment.

We report a genome-wide, multiscale approach to simultaneously measure the effect that the increased copy of each gene and/or operon has on a desired trait or phenotype. The method involves (i) growth selections on a mixture of several different plasmid-based genomic libraries of defined insert sizes or SCALEs, (ii) microarray studies of enriched plasmid DNA, and a (iii) mathematical multiscale analysis that precisely identifies the relevant genetic elements. This approach allows for identification of all single open reading frames and larger multigene fragments within a genomic library that alter the expression of a given phenotype. We have demonstrated this method in Escherichia coli by monitoring, in parallel, a population of >10(6) genomic library clones of different insert sizes, throughout continuous selections over a period of 100 generations.

DNA Fragmentation↗
PubMed2014

Engineering adeno-associated viruses for clinical gene therapy.

Clinical gene therapy has been increasingly successful owing both to an enhanced molecular understanding of human disease and to progressively improving gene delivery technologies. Among these technologies, delivery vectors based on adeno-associated viruses (AAVs) have emerged as safe and effective and, in one recent case, have led to regulatory approval. Although shortcomings in viral vector properties will render extension of such successes to many other human diseases challenging, new approaches to engineer and improve AAV vectors and their genetic cargo are increasingly helping to overcome these barriers.

Capsid↗
PubMed2002

A new method for the preparation of human parathyroid hormone 1-34 peptides.

An engineered Escherichia coli strain, BL21 (DE3)/pGEX-4T-human parathyroid hormone (hPTH) (1-34), was constructed by oligonucleotide annealing and PCR amplification of the target gene, and then by ligating it with the pGEX-4T-3 vector and transferring into the BL21 host. The soluble glutathione S-transferase (GST) fusion protein GST-hPTH (1-34), expressed from BL21 (DE3)/pGEX-4T-hPTH (1-34), was harvested after fermentation and purification by affinity chromatography. Following double cleavage by thrombin and prolyl endopeptidase, about 0.6 g/l intact hPTH (1-34) was harvested. The product was checked by HPLC MS and N-terminal sequence analysis. The purified recombinant hPTH (1-34) stimulates adenylate cyclase in rabbit renal cortical cell membranes to exactly the same extent as synthetic hPTH standards, indicating that the recombinant product has full biological activity.

Adenylyl Cyclases↗
PubMed2006

Differential analysis of secondary metabolites by LC-MS following strain improvement of Streptomyces lydicus AS 4.2501.

Metabolite variations in a high-yielding mutant and its parent strain were studied by comparative LC-MS analysis after strain improvement. Streptomyces lydicus AS 4.2501-P28, a propionate-resistant mutant isolated by the high-frequency screening method using the principle of eliminating precursor inhibition effects, showed an increase of 267% in streptolydigin titre over the starting strain. Culture extracts of this mutant and its parent strain were analysed in parallel by an LC-MS technique, including full scan and extracted-ion scan, ESI-MS (electrospray-ionization MS) detection, DAD (diode-array detection) and MS2 (tandem MS) measurement. The main metabolic variations were obviously found in intermediates, metabolites and biosynthetic pathways: two unknown metabolites with the molecular [M-H]- ions at m/z 423.3 and 687.2, corresponding to two branch pathways, were blocked in the mutant, and the accumulation of a significant intermediate at m/z 363.1 [M-H]- decreased dramatically in the mutant cultures, resulting in the overproduction of streptolydigin (an antibiotic that inhibits prokaryotic RNA polymerase) in the mutant. Ion fragmentations of the tandem-MS spectra provided experimental evidence for the structural characterization of the three compounds obtained. In comparison with the traditional methods, comparative LC-MS analysis was rapid, sensitive and suitable for characterizing intermediates, metabolites and pathways for elucidation of the metabolic alterations after the isolation of improved strains.

Aminoglycosides↗
PubMed2002

Generation of Campylobacter jejuni genetic diversity in vivo.

Molecular epidemiology studies suggest that horizontal genetic exchange is a major cause of pathogen biodiversity. We tested this concept for the bacterial enteropathogen Campylobacter jejuni by seeking direct in vivo evidence for the exchange of genetic material among Campylobacter strains. For this purpose, two antibiotic resistance markers were inserted into the hipO or htrA gene of genetically distinct and naturally transformable C. jejuni strains. Genetic exchange of the resistance markers was analysed after co-cultivation of homologous and heterologous strains in vitro and in vivo during experimental infection of chickens. Double-resistant recombinants were obtained both in vitro and from the chicken intestine for all combinations of strains tested. Bidirectional genetic exchange of DNA between homologous and heterologous strains was confirmed by Southern blotting in combination with flaA polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE). Extensive PFGE analyses of isolated recombinants indicated the frequent occurrence of genetic rearrangements during the experimental infection, in addition to the homologous recombination of the antibiotic resistance genes. Together, the data indicate unequivocally that interstrain genetic exchange as well as intragenomic alterations do occur in vivo during C. jejuni infection. These events probably explain the genome plasticity observed for this pathogen.

Animals↗

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