The pGEX-3x plasmid is a popular cloning vector.

A cloning vector is a small DNA vehicle that carries a foreign DNA fragment. The insertion of the fragment into the cloning vector is carried out by treating the vehicle and the foreign DNA with the same restriction enzyme, then ligating the fragments together. There are many types of cloning vectors. Plasmids and bacteriophages (such as phage λ) are perhaps most commonly used for this purpose. Other types of cloning vectors include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Wikipedia does not have an article with this exact name. ... Wikipedia does not have an article with this exact name. ... The general structure of a section of DNA Deoxyribonucleic acid (DNA) is a nucleic acid —usually in the form of a double helix— that contains the genetic instructions specifying the biological development of all cellular forms of life, and most viruses. ... A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases. ... In biochemistry, a ligase is an enzyme that can catalyse the joining of two molecules (ligation or gluing together) by forming a new chemical bond, with accompanying hydrolysis of ATP or other similar molecules. ... Figure 1: Schematic drawing of a bacterium with plasmids enclosed. ... A phage (also called bacteriophage) (in Greek phageton = food/consumption) is a small virus that infects only bacteria. ... A bacterial artificial chromosome (BAC) is a DNA construct, based on a fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. ... A yeast artificial chromosome (short YAC) is a vector used to clone large DNA fragments (up to 400 kb). ...

Common Features

Most commercial cloning vectors have a couple of key features that have made their use in molecular biology so widespread. Molecular biology is the study of biology at a molecular level. ...

Usually, the main purpose of cloning vector is the controlled expression of a particular gene inside a convenient host organism (eg. E. coli). Control of expression can be very important; it is usually desirable to insert the target DNA into a site that is under the control of a particular promoter. Some commonly used promoters are T7 promoters, lac promoters (bla promoter) and cauliflower mosaic virus's 35s promoter (for plant vectors). To allow for convenient and favorable insertions, most cloning vectors have had nearly all their restriction sites engineered out of them and a multiple cloning site (MCS) inserted that contains many restriction sites. MCSs allow for insertions of DNA into the vector to be targeted and possibly directed in a chosen orientatiton. A selectable marker, such as antibiotic resistance [eg. beta-lactamase (see figure)] is often included in the vector to identify postively transformed cells. All inserted DNA (plasmids etc.) need an origin of replication (ORI; not shown in figure). High strigency ORIs are preferable for cloning vectors. Binomial name Escherichia coli T. Escherich, 1885 Escherichia coli (usually abbreviated to E. coli) is one of the main species of bacteria that live in the lower intestines of warm-blooded animals (including birds and mammals) and are necessary for the proper digestion of food. ... The general structure of a section of DNA Deoxyribonucleic acid (DNA) is a nucleic acid —usually in the form of a double helix— that contains the genetic instructions specifying the biological development of all cellular forms of life, and most viruses. ... In genetics, a promoter is a DNA sequence that enables a gene to be transcribed. ... Bacteriophage T7 Capsid size The T7 capsid is spherical with an inner diameter of 55 nm. ... The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. ... Plant viruses are viruses affecting plants. ... A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases. ... Also known as an MCS, a multiple cloning site is a short segment of DNA which contains many (usually 10+) restriction sites - a standard feature of engineered plasmids. ... An antibiotic is a drug that kills or slows the growth of bacteria. ... Beta-lactamase is a type of enzyme (EC 3. ... Figure 1 : Schematic drawing of a bacterium with plasmids enclosed. ... The origin of replication (also called the replication origin) is a unique DNA sequence at which DNA replication is initiated. ...

Some other possible features of cloning vectors are: vir genes for plant transformation, intergrase sites for chromosomal insertion, lac Z alpha fragment for blue-white selection, and/or in-frame genes attached to the MCS for recombinant proteins [eg. Green fluorescent protein (GFP) or glutathione S-transferase (see figure)]. Species Agrobacterium tumefaciens Agrobacterium rhizogenes Agrobacterium is a genus of bacteria that cause tumors in plants. ... Transposons are sequences of DNA that can move around to different positions within the genome of a single cell, a process called Transposition. ... The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. ... Also known as an MCS, a multiple cloning site is a short segment of DNA which contains many (usually 10+) restriction sites - a standard feature of engineered plasmids. ... Recombinant proteins are proteins that are produced by different genetically modified organisms following insertion of the relevant DNA into their genome. ... GFP ribbon diagram from PDB database The green fluorescent protein (GFP) is a protein from the jellyfish Aequorea victoria that fluoresces green when exposed to blue light. ... The Glutathione S-transferase (GST) family of enzymes comprises a long list of cytosolic, mitochondrial, and microsomal proteins which are capable of multiple reactions with a multitude of substrates, both endogenous and xenobiotic. ...

SCREENING: BLUE AND WHITE SELECTION General purpose vectors such as pUC19 usually include a system for detecting the prescence of a cloned DNA fragment, based on the loss of an easily scored phenotype. The most widely used system uses the lac z system encoding for beta galactosidase in e coli.Active beta galactosidase can be detected by its ability to change the substrate x gal (5 bromo-4-chloro-3-indolyl-beta-d-galactoside) from colourless to blue. Cloning a fragment of DNA within the vector based gene encoding the beta galactosidase prevents the formation of an active beta galactosidase. If x gal is included in the selective agar plates, transformant colonies are blue in the case of a vector with no inserted DNA and white in the case of a vector containing a fragment of cloned DNA.