Recombinant DNA Recombinant DNA Technology Technology

Biochemistry, 2005

The modern science mainly treats the biochemical basis of sequencing in bio-macromolecules and processes in medicine and biochemistry. One can ask weather the language of biochemistry is the adequate scientific language to explain the phenomenon in that science. Is there maybe some other language, out of biochemistry, that determines how the biochemical processes will function and what the structure and organization of life systems will be? The research results provide some answers to these questions. They reveal to us that the process of sequencing in bio-macromolecules is conditioned and determined not only through biochemical, but also through cybernetic and information principles. Many studies have indicated that analysis of protein sequence codes and various sequencebased prediction approaches, such as predicting drug-target interaction networks [14], predicting functions of proteins [15,18], analysis and prediction of the metabolic stability of proteins [16], predicting the network of substrate-enzymeproduct triads [7], membrane protein type prediction [1,2,5]. protein structural class prediction [4,12], protein secondary structure prediction [6,11], enzyme family class prediction [3,11], identifying cyclin proteins [20], protein subcellular location prediction [9,10,17,19] , among many others as summarized in a recent review [15] , can timely provide very useful information and insights for both basic research and drug design and hence are widely welcome by science community. The present study is attempted to develop a novel sequence-based method for studying insulin in hopes that it may become a useful tool in the relevant areas.

Background: Standard biological parts, such as BioBricks™ parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks™ assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon. Results: Here, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome. Conclusions: The BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.

▲ FIGURE 4-2 Alternative representations of a nucleic acid strand illustrating its chemical directionality. Shown here is a single strand of DNA containing only three bases: cytosine (C), adenine (A), and guanine (G). (a) The chemical structure shows a hydroxyl group at the 3Ј end and a phosphate group at the 5Ј end. Note also that two phosphoester bonds link adjacent nucleotides; this two-bond linkage commonly is referred to as a phosphodiester bond. (b) In the "stick" diagram (top), the sugars are indicated as vertical lines and the phosphodiester bonds as slanting lines; the bases are denoted by their single-letter abbreviations. In the simplest representation (bottom), only the bases are indicated. By convention, a polynucleotide sequence is always written in the 5Јn3Ј direction (left to right) unless otherwise indicated. 287:755; part (b) from R. E. Dickerson, 1983, Sci. Am. 249:94.]

Electronic Journal of Biotechnology, 2006

Aim: To isolate the genomic DNA from E .coli DH5α cells.

A synthetic operon was constructed using the reporter genes gfp and lacZ and the arabinose-inducible araBAD promoter. DNA cassettes encoding mRNA secondary structures were placed at the 3′ and 5′ ends of the genes and a putative RNase E site was placed between the genes. These mRNA control elements have been shown to affect transcript processing and decay, resulting in altered protein levels. These constructs were transformed into cells harboring the native arabinose-inducible araE gene encoding the arabinose transport protein and engineered cells harboring a constitutively expressed araE. In the strains with arabinose-dependent transport the linear response in the production of both reporter proteins to inducer concentration occurred over a narrow range of arabinose concentrations. In the strains with constitutive transport the linear range of gene expression occurred over a much larger arabinose concentration range than in strains with the arabinose-inducible transport. Strains with the arabinose-inducible transport harboring different operon constructs produced the two reporter proteins at very different levels at low arabinose concentrations; as inducer concentrations increased, differences in relative expression levels decreased. In contrast, strains with constitutive transport harboring different operon constructs produced the reporter proteins at very different levels across the entire range of inducer concentrations, pointing to the importance of optimizing gene expression control at various levels to control the production of heterologous proteins.

2011

PART I: FOUNDATIONS Studying the Molecules of Life DNA: The Repository of Biological Information Chemical Basis of Information Molecules Protein Structure Protein Function PART II: NUCLEIC ACID STRUCTURE & METHODS DNA and RNA Structure Studying Genes Genomes, Transcriptomes, and Proteomes Topology: Functional Deformations of DNA Nucleosomes, Chromatin, and Chromosome Structure PART III: INFORMATION TRANSFER DNA Replication DNA Mutation and Repair Moment of Discovery Homologous Recombination Site-Specific Recombination and Transposition DNA-Dependent Synthesis of RNA RNA Processing The Genetic Code Protein Synthesis PART IV: REGULATION Regulating the Flow of Information The Regulation of Gene Expression in Bacteria Transcriptional Regulation of Gene Expression in Eukaryotes Post-Transcriptional Regulation of Gene Expression in Eukaryotes Gene Regulation in Development Online Appendix: Model Organisms