PCR-based engineering of yeast genome.
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DNA Primers↗
SEARCH · PubMed Health
Results for “Genome engineering”
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Designing better phages.
We propose a method to engineer the genome of bacteriophages to increase their effectiveness as antibacterial agents. Specifically, we exploit the redundancy of the triplet code to design genomes that avoid restriction sites while producing the same proteins as wild-type phages. We give an efficient algorithm to minimize the number of restriction sites against sets of cutter sequences, and demonstrate that that phage genomes can be significantly protected against surprisingly large sets of enzymes with no loss of function. Finally, we develop a model to explain why evolution has failed to eliminate many possible restriction sites despite selective pressure, thus motivating the need for genome-level sequence engineering.
Algorithms↗
Natural genetic engineering of the bacterial genome.
The term 'natural genetic engineering' means viewing genetic change as a coordinated cell biological process, the reorganization of discrete genomic modules, resulting in the formation of new DNA structures. Examples of natural genetic engineering continue to accumulate, and the concept can be used to integrate observations which demonstrate the similarity between in vitro genetic engineering and the action of in vivo agents of genetic change.
DNA, Bacterial↗
Engineering the plastid genome of higher plants.
The plastid genome of higher plants is an attractive target for engineering because it provides readily obtainable high protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs and gene containment through the lack of pollen transmission. A chloroplast-based expression system that is suitable for the commercial production of recombinant proteins in tobacco leaves has been developed recently. This expression system includes vectors, expression cassettes and site-specific recombinases for the selective elimination of marker genes. Progress in expressing proteins that are biomedically relevant, in engineering metabolic pathways, and in manipulating photosynthesis and agronomic traits is discussed, as are the problems of implementing the technology in crops.
Crops, Agricultural↗
Genetic engineering of novel genomes of large DNA viruses.
Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals.
Base Sequence↗
[Genome structure, molecular genetics and genetic engineering of cyanobacteria].
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Chromosome Mapping↗
Constructing an efficient trans-acting genomic HDV ribozyme.
We have engineered a genomic HDV ribozyme to construct several trans-acting ribozymes for use in trans to cleave target RNAs. Among the 10 different combinations attempted, only HDV88-Trans had cleavage activity on the 13-nucleotide substrate, R13, in vitro. To improve the cleavage efficiency, at least in vitro, of the HDV88-Trans ribozyme (kclv = 0.022 min(-1)), we have constructed several variants that differ in forming stem II (length) in the pseudoknot secondary structure model. When cleavage rate constants were analyzed and compared among variants of HDV88-Trans, HDV88-Trans-4 yielded kclv = 1.7 min(-1). HDV88-Trans-4 thus represents the highest active genomic HDV ribozyme that functions in trans thus far constructed, and has activity under physiological conditions (pH 7.1 at 37 degrees C with 1 mM of MgCl2).
Base Sequence↗
Genetic engineering, post-genomic ethics, and the Catholic tradition: ethical overview.
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Animals↗
Engineering the mouse genome by site-specific recombination.
Site-specific recombination systems are powerful tools for introducing predetermined modifications into eukaryotic genomes. Recent advances allow the manipulation of chromosomal DNA in a spatially and temporally controlled manner in mice, offering unprecedented possibilities for studying mammalian genome function and for generating animal models for human diseases.
Animals↗
Genomic instability--the engine of tumorigenesis?
Human cancers harbour numerous mutations and it has been proposed that these result from some form of inherent genomic instability. Some cancers have proven genomic instability or features that are indicative of this. Inherited cancer syndromes exist that are caused by deficient DNA repair or chromosomal integrity. By contrast, theoretical analysis and experimental data from sporadic colorectal tumours provide little general evidence of genomic instability in early lesions. These apparently conflicting data raise the question of whether or not genomic instability is necessary for driving tumour growth, and whether or not it is the usual initiating event in tumorigenesis.
Cell Transformation, Neoplastic↗
New vectors and marker excision systems mark progress in engineering the plastid genome of higher plants.
The transformation of the plastid genome, until recently restricted to tobacco, is now being extended to a rapidly growing list of crops. This perspective provides an overview of emerging trends of technology development in the field with a focus on vector design and marker excision systems. The new tools will facilitate engineering of the photosynthetic machinery and enable novel agricultural and industrial applications.
Genes, Reporter↗
Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.
Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrBF267D ) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaXL289Q ) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.
Escherichia coli↗
Plasticity of the streptomyces genome-evolution and engineering of new antibiotics.
Streptomyces is a genus of soil dwelling bacteria with the ability to produce natural products that have found widespread use in medicine. Annotation of Streptomyces genome sequences has revealed far more biosynthetic gene clusters than previously imagined, offering exciting possibilities for future combinatorial biosynthesis. Experiments to manipulate modular biosynthetic clusters to create novel chemistries often result in no detectable product or product yield is extremely low. Understanding the coupling between components in these hybrid enzymes will be crucial for efficient synthesis of new compounds. We are using new algebraic approaches to predict protein properties, and homologous recombination to exploit natural evolutionary constraints to generate novel functional enzymes. The methods and techniques developed could easily be adapted to study modular, multi-interacting complex systems where appreciable biochemical and comparative sequence data are available, for example, clinically significant non-ribosomally synthesised peptides and polyketides.
Anti-Bacterial Agents↗
Genome organization, natural genetic engineering and adaptive mutation.
Bacterial evolution is considered in the light of molecular discoveries about genome organization, biochemical mechanisms of genetic change, and cellular control networks. Prokaryotic genetic determinants are organized as modular composites of coding sequences and protein-factor binding sites joined together during evolution. Studies of genetic change have revealed the existence of biochemical functions capable of restructuring the bacterial genome at various levels and joining together different sequence elements. These natural genetic engineering systems can be subject to regulation by signal transduction networks conveying information about the extracellular and intracellular environments. Mu-mediated araB-lacZ coding sequence fusions provide one example of adaptive mutation (increased formation of useful mutations under selection) and illustrate how physiological regulation can modulate the activity of a natural genetic engineering system under specific conditions.
Bacteria↗
The emerging impact of genomics on the development of biological weapons. Threats and benefits posed by engineered extremophiles.
During the past decades, representatives of Archaea, Bacteria, and Protista have been found thriving in many newly discovered extremely hostile habitats, which hitherto were regarded as too harsh to harbor life. To illustrate how an extremophile could be targeted for development as a biowarfare agent, an example is presented describing current advances in engineering Deinococcus radiodurans. Using a generally applicable combination of conventional genetic engineering and genomic informatics, this extremely radiation-resistant and environmentally robust bacterium is being developed for biotechnology.
Archaea↗
Impact of 'ome' analyses on inverse metabolic engineering.
Genome-wide or large-scale methodologies employed in functional genomics such as DNA sequencing, transcription profiling, proteomics, and metabolite profiling have become important tools in many metabolic engineering strategies. These techniques allow the identification of genetic differences and insight into their cellular effects. In the field of inverse metabolic engineering mapping of differences between strains with different degree of a certain desired phenotype and subsequent identification of factors conferring that phenotype are an essential part. Therefore, the tools of functional genomics in particular have the potential to promote and expand inverse metabolic engineering. Here, we review the use of functional genomics methods in inverse metabolic engineering, examples are presented, and we discuss the identification of targets for metabolic engineering with low fold changes using these techniques.
Animals↗
Framework for a fully powered risk engine.
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Genome, Human↗
Engineering the chloroplast genome: techniques and capabilities for chloroplast transformation in Chlamydomonas reinhardtii.
Chloroplast transformation of Chlamydomonas reinhardtii has been accomplished by agitating cell wall-deficient cells in the presence of glass beads and DNA. By using the atpB gene as the selected marker and cells grown in 0.5 mM 5-fluorodeoxyuridine, we have recovered up to 50 transformants per microgram of DNA. This method is easy and does not require specialized equipment, although it is not as efficient as the tungsten particle bombardment method [Boynton, J. E., Gillham, N. W., Harris, E. H., Hosler, J. P., Johnson, A. M., Jones, A. R., Randolph-Anderson, B. L., Robertson, D., Klein, T. M., Shark, K. B. & Sanford, J. C. (1988) Science 240, 1534-1537]. By using particle bombardment, we have developed a cotransformation approach in which spectinomycin-resistant 16S rRNA-encoding DNA is the selected marker, and we have demonstrated that cotransformation of an unselected marker on an independent replicon is very efficient. We have used this strategy (i) to recover transformants with partially deleted atpB genes that could not otherwise have been selected since they did not restore photosynthetic capability to a recipient carrying a more extensive atpB deletion and (ii) to generate specific deletion mutations in a wild-type recipient. This methodology should allow the introduction of any desired change into the chloroplast genome, even in the absence of phenotypic selection, and thus a detailed functional analysis of any chloroplast DNA sequence should be possible.
Journal Article↗