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Expression Cloning
Expression cloning is a technique in DNA cloning that uses expression vectors to generate a library of clones, with each clone expressing one protein. This ''expression library'' is then screened for the property of interest and clones of interest are recovered for further analysis. An example would be using an expression library to isolate genes that could confer antibiotic resistance. Expression vectors Expression vectors are a specialized type of cloning vector in which the transcriptional and translational signals needed for the regulation of the gene of interest are included in the cloning vector. The transcriptional and translational signals may be synthetically created to make the expression of the gene of interest easier to regulate. Purpose Usually the ultimate aim of expression cloning is to produce large quantities of specific proteins. To this end, a bacterial expression clone may include a ribosome binding site ( Shine-Dalgarno sequence) to enhance translation of ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
DNA Cloning
Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word ''cloning'' refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine. In a conventional molecular cloning experiment, the DNA to be cloned is obtained from an organism of interest, then treated with enzymes in the test tube to generate smaller DNA fragments. Subsequently, these fragments are then combined with vector DNA to generate recombinant DNA molecules. The recombinant DNA is then in ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Transcription (genetics)
Transcription is the process of copying a segment of DNA into RNA. The segments of DNA transcribed into RNA molecules that can encode proteins are said to produce messenger RNA (mRNA). Other segments of DNA are copied into RNA molecules called non-coding RNAs (ncRNAs). mRNA comprises only 1–3% of total RNA samples. Less than 2% of the human genome can be transcribed into mRNA ( Human genome#Coding vs. noncoding DNA), while at least 80% of mammalian genomic DNA can be actively transcribed (in one or more types of cells), with the majority of this 80% considered to be ncRNA. Both DNA and RNA are nucleic acids, which use base pairs of nucleotides as a complementary language. During transcription, a DNA sequence is read by an RNA polymerase, which produces a complementary, antiparallel RNA strand called a primary transcript. Transcription proceeds in the following general steps: # RNA polymerase, together with one or more general transcription factors, binds to promoter DNA ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
PBR322
pBR322 is a plasmid and was one of the first widely used '' E. coli'' cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdoctoral researcher and Raymond L. Rodriguez. The p stands for "plasmid," and BR for "Bolivar" and "Rodriguez." pBR322 is 4361 base pairs in length and has two antibiotic resistance genes – the gene '' bla'' encoding the ampicillin resistance (AmpR) protein, and the gene ''tetA'' encoding the tetracycline resistance (TetR) protein. It contains the origin of replication of pMB1, and the '' rop'' gene, which encodes a restrictor of plasmid copy number. The plasmid has unique restriction sites for more than forty restriction enzymes. Eleven of these forty sites lie within the TetR gene. There are two sites for restriction enzymes HindIII and ClaI within the promoter of the TetR gene. There are six key restriction sites inside the AmpR gene.Th ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
λ Phage
''Enterobacteria phage λ'' (lambda phage, coliphage λ, officially ''Escherichia virus Lambda'') is a bacterial virus, or bacteriophage, that infects the bacterial species ''Escherichia coli'' (''E. coli''). It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell. The phage particle consists of a head (also known as a capsid), a tail, and tail fibers (see image of virus below). The head contains the phage's double-strand linear DNA genome. During infection, the phage particle recognizes and binds to its host, ''E. coli'', causing DNA in the head of the phage to be ejected through the tail into t ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Translation (biology)
In molecular biology and genetics, translation is the process in which ribosomes in the cytoplasm or endoplasmic reticulum synthesize proteins after the process of transcription (biology), transcription of DNA to RNA in the cell's nucleus (cell), nucleus. The entire process is called gene expression. In translation, mRNA, messenger RNA (mRNA) is decoded in a ribosome, outside the nucleus, to produce a specific amino acid chain, or polypeptide. The polypeptide later protein folding, folds into an Activation energy, active protein and performs its functions in the Cell (biology), cell. The ribosome facilitates decoding by inducing the binding of Base pair, complementary tRNA anticodon sequences to mRNA codons. The tRNAs carry specific amino acids that are chained together into a polypeptide as the mRNA passes through and is "read" by the ribosome. Translation proceeds in three phases: # Initiation: The ribosome assembles around the target mRNA. The first tRNA is attached a ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Gene Expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product that enables it to produce end products, protein or non-coding RNA, and ultimately affect a phenotype, as the final effect. These products are often proteins, but in non-protein-coding genes such as transfer RNA (tRNA) and small nuclear RNA (snRNA), the product is a functional non-coding RNA. Gene expression is summarized in the central dogma of molecular biology first formulated by Francis Crick in 1958, further developed in his 1970 article, and expanded by the subsequent discoveries of reverse transcription and RNA replication. The process of gene expression is used by all known life—eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea), and utilized by viruses—to generate the macromolecular machinery for life. In genetics, gene expression is the most fundamental level at which the genotype gives rise to the phenotype, '' ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Genetics
Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinian friar working in the 19th century in Brno, was the first to study genetics scientifically. Mendel studied "trait inheritance", patterns in the way traits are handed down from parents to offspring over time. He observed that organisms (pea plants) inherit traits by way of discrete "units of inheritance". This term, still used today, is a somewhat ambiguous definition of what is referred to as a gene. Trait inheritance and molecular inheritance mechanisms of genes are still primary principles of genetics in the 21st century, but modern genetics has expanded to study the function and behavior of genes. Gene structure and function, variation, and distribution are studied within the context of the cell, the organism (e.g. dominance), and within the ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Posttranslational Modification
Post-translational modification (PTM) is the covalent and generally enzymatic modification of proteins following protein biosynthesis. This process occurs in the endoplasmic reticulum and the golgi apparatus. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. PTMs are important components in cell signal transduction, signaling, as for example when prohormones are converted to hormones. Post-translational modifications can occur on the amino acid side chains or at the protein's C-terminus, C- or N-terminus, N- termini. They can extend the chemical repertoire of the 20 standard amino acids by modifying an existing functional group or introducing a new one such as phosphorylation, phosphate. Phosphorylation is a highly effective mechanism for regulating the activity of enzymes and is the most common post-translational modification. Many eukaryotic and prokaryotic proteins also have carbohydra ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Eukaryotes
Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bacteria and Archaea (both prokaryotes) make up the other two domains. The eukaryotes are usually now regarded as having emerged in the Archaea or as a sister of the Asgard archaea. This implies that there are only two domains of life, Bacteria and Archaea, with eukaryotes incorporated among archaea. Eukaryotes represent a small minority of the number of organisms, but, due to their generally much larger size, their collective global biomass is estimated to be about equal to that of prokaryotes. Eukaryotes emerged approximately 2.3–1.8 billion years ago, during the Proterozoic eon, likely as flagellated phagotrophs. Their name comes from the Greek εὖ (''eu'', "well" or "good") and κάρυον (''karyon'', "nut" or "kernel"). Eu ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Expression Vector
An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins. The vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the efficient production of protein, and this may be achieved by the production of significant amount of stable messenger RNA, which can then be translated into protein. The expression of a protein may be tightly controlled, and the protein is only produced in significant quantity when necessary through the use of an inducer, in some systems however the protein ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
Binding Site
In biochemistry and molecular biology, a binding site is a region on a macromolecule such as a protein that binds to another molecule with specificity. The binding partner of the macromolecule is often referred to as a ligand. Ligands may include other proteins (resulting in a protein-protein interaction), enzyme substrates, second messengers, hormones, or allosteric modulators. The binding event is often, but not always, accompanied by a conformational change that alters the protein's function. Binding to protein binding sites is most often reversible (transient and non-covalent), but can also be covalent reversible or irreversible. Function Binding of a ligand to a binding site on protein often triggers a change in conformation in the protein and results in altered cellular function. Hence binding site on protein are critical parts of signal transduction pathways. Types of ligands include neurotransmitters, toxins, neuropeptides, and steroid hormones. Binding sites in ... [...More Info...] [...Related Items...] OR: [Wikipedia] [Google] [Baidu] |
