The restriction fragment was inserted into pbr322 via these bamhi and ecori sites and cloned in. Plasmid vector pbr322 and its specialpurpose derivativesa. Ampicillin versus tetracycline in the selection of pbr322. A vector is used to amplify a single molecule of dna into many copes. The plasmid pbr322 was one of the first ek2 multipurpose cloning vectors to be. The pbr322 plasmid is a commonly used cloning vector that contains both the ampicillin and tetracycline resistance genes as selectable markers.
The plasmid pbr322 was stably maintained under all growth conditions tested. It is derived from pbr322 by insertion into its ecor i site of the chloramphenicol acetyltransferase gene. The molecular analysis of dna has been made possible by the cloning of dna. This chapter explains the process of inserting cloning into puc and discusses the growth of plasmids. The plasmid pbr322 was constructed by a series of in uiuo and in uitro ligation events, with the explicit aim of generating a multipurpose cloning vector. Inserts cloned in this vector may be characterized easily using this data.
Because a number of unique cloning sites are available in the polylinker region of puc vectors, the puc plasmid is often a more versatile subcloning vector than pbr322. An isolated dna fragment from a eukaryotic genome also produced by psti cleavage is added to the prepared vector and ligated. These fragments can serve as accurate dna size markers from small pieces up to the 4362 base pair length of pbr322. Plasmid cloning vectors 181 has proved to be the first of a large and continuouslyevolvingfamily of vectors balbas et af. One example of a plasmid used for dna cloning is called pbr322 plasmid. This page is informational only this vector is not available from addgene please contact the manufacturer for further details. To use pbr322 plasmid to clone a gene, a restriction endonuclease first cleaves the plasmid at a restriction site. Cloning vectorcharacteristics and types online biology. Pbr322 pbr322 low copy cloning vector plasmid vector for. Pbr322 pbr322 low copy cloning vector plasmid vector. It is a prokaryotic vector carrying a multiple cloning site with unique restriction sites. The mixture of ligated dnas is then used to transform bacteria, and plasmidcontaining bacteria are selected by growth. This vector was small, 4 kb in size, and had two antibiotic resistance genes for selection. Cloning vector characteristics and types cloning vector.
As a starting material for cloning of casein gene sequences. The molecule is a doublestranded circle 4,361 base pairs in length 2. The cloning vector may be dna taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium. Pbr322 pbr322 low copy cloning vector plasmid vector for molecular cloning. Derived from pbr322 by deleting sequences between nt 1430 avai and 2519, thus removing the bom or. The plasmid pbr322 was one of the first ek2 multipurpose cloning vectors to be designed and constructed ten years ago for the efficient cloning and selection. It is one of the most widely used plasmid for cloning in li. The plasmids are grown under conditions identical to those of pbr322. A cloning vector is a small piece of dna that can be stably maintained in an organism, and into which a foreign dna fragment can be inserted for cloning purposes.
Convenient expression of cloned genes in vitro or in vivo. Construction of a recombinant bacterial plasmid containing a chick. Promoter p1 is artificially created by the ligation of two different dna fragments to create pbr322. The puc18 vector was isolated from li top10 strain. Structural biochemistrydna recombinant techniquesplasmid. The p stands for plasmid, and br for bolivar and rodriguez. The two molecules that are required for cloning are the dna to be cloned and a cloning vector. The pmb1 of puc18 differs from the pbr322 origin by a single point mutation and the. Cloning vector a dna molecule that carries foreign dna into a host cell, replicates inside a. Pcr cloning vectors with 3 options for insert excision. The molecule is a doublestranded circle 4,361 base pairs in length. In 1977, they described the first vector designed for cloning purposes, pbr322 a plasmid. Insertion of this fragment into a modified version of pbr322 results in an amprtetr vector pjrd158 of 3903 bp containing 28 unique cloning sites.
Plasmid pbr322 is one of the most widely employed cloning vehicles 1. Promoter p3 is the natural promoter for the betalactamase gene. Plasmid subcloning vector that carries genes for tetracycline and ampicillin. 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 who constructed it. It is unclear, if this is a mutation in pmac58, or if the sequence of pbr325 is incorrect at that position. It is approximately 4300 bp in length and has two antibiotic resistance genes. The amp r gene originally residing on plasmid r1, tet r from r65. The plasmid pbr322 was one of the first ek2 multipurpose cloning vectors to be designed and constructed ten years ago for the efficient cloning and selection of recombinant dna molecules in. Plasmids pbr322 and puc8 pbr322 plasmid one of the first plasmids to be used in recombinant genetics was called pbr322. The plasmid pbr325 is a cloning vector constructed in vitro by the addition of the chloramphenicol resistance gene of an is1flanked transposon to pbr322. A cloning vector is a genome that can accept the target dna and increase the number of copies through its own autonomous replication. It has been constructed using the ampicillin resistance gene and the pmb1 origin of replication from pbr322. Maintenance and genetic stability of vector plasmids.
The exact sizes of all restriction fragments and the relative positions of the cuts are presented. Balbas p, soberon x, merino e, zurita m, lomeli h, valle f, flores n, bolivar f. Plasmid pbr322 1 contains three unique restriction endonuclease recognition sites within the. The rop gene product, which regulates plasmid replication by stabilizing the interaction between rnai. The most widely used of the early purposebuilt vectors is pbr322. Created in 1977 in the laboratory of herbert boyer at the university of california.
The vector therefore contains features that allow for the convenient insertion or removal of a dna. The pbr322 plasmid contains a gene that allow the bacteria to be resistant to the antibiotics tetracycline and amipicillin. The replication origin of pbr322, which directs multiplication of the cloning vector in host cells is originally from pmb1. Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. Cloning vector is used for replicating donor dna fragment within host cell.
This plasmid vector has been put together from fragments originating from three different naturally. This vector comes readytouse for enzymatic reactions and transformations. A repository of over 200,000 plasmids including protein structure initiative protein expression plasmids and vectors, over 75,000 human plasmids, and. Function of ori, restriction enzyme and selectable markers explained. The widespread use of pbr322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmids effectiveness as a cloning vector. This is a free resource for the scientific community that is compiled by addgene. Thermo scientific pbr322 is one of the most commonly used li cloning vectors. The plasmid cloning vector pbr322, shown here, is cleaved with the restriction endonuclease psti. Cloning vectors cloning vectors are dna molecules that are used to transport cloned sequences between biological hosts and the test tube.
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