Plasmid vector pbr322 and its specialpurpose derivatives. 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. Six ml of cells were collected by centrifugation at 2500 rpm for 5 min at 4 c. Ampicillin versus tetracycline in the selection of pbr322. Pbr322 pbr322 low copy cloning vector plasmid vector. Among these, pbr322 has been completely sequenced through modification of earlier plasmids of e. Plasmid and vector are two types of doublestranded dna molecules that have different functions in the cell.
The cells were resus pended in 210 pl of cold 50 mm trisc1, ph 7. The cole1type plasmid pbr322 is commonly used as vector tool in molecular biology. It is approximately 4300 bp in length and has two antibiotic resistance genes. The rop gene product, which regulates plasmid replication by stabilizing the interaction between rnai and rnaii transcripts, maintains the copy number at about 20 per cell. The phenotype of bacteria containing the intact plasmid is amp r tet r. The mixture of ligated dnas is then used to transform bacteria, and plasmidcontaining bacteria are selected by growth. Plasmids pbr322 and puc8 pbr322 plasmid one of the first plasmids to be used in recombinant genetics was called pbr322. Promoter p2 in the same region as p1, but it is on the opposite strand and initiates transcription in the direction of the tetracycline resistance gene. Insertion of this fragment into a modified version of pbr322 results in an amprtetr vector pjrd158 of 3903 bp. Results from a previous experiment indicate that different selective. The pbr322 vector 4,361bp carries the genes for tetracycline and ampicillin resistance. Electron microscopic analysis of in vitro transcriptional complexes of pbr322 and pacyc184 revealed five and six major transcriptional units, respectively, in these two plasmid vectors. An isolated dna fragment from a eukaryotic genome also produced by psti cleavage is added to the prepared vector and ligated.
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. Best trick trick for pbr322 ncert genetics youtube. Function of ori, restriction enzyme and selectable markers explained. Plasmid cloning vectors 181 has proved to be the first of a large and continuouslyevolvingfamily of vectors. Restriction analysis of plasmid dna in this exercise, you will digest the plasmid pbr322 with 5 different restriction enzymes and resolve the fragments by agarose gel electrophoresis.
Plasmid subcloning vector that carries genes for tetracycline and ampicillin. The antibioticresistant genes on pbr322 are not transposable. In cloning experiments, the pbr group of plasmids is the most widely used cloning vectors. This plasmid contains a replication origin, several recognition sequences for different restriction enzymes with names like ecori, and two marker genes, which confer resistance to different antibiotics ampicillin and tetracycline electron microscopic analysis of in vitro transcriptional complexes of pbr322. It contains three unique restriction endonuclease recognition sites within the. The cosmid vector can carry up to 45 kb whereas plasmid and. The pbr322 plasmid is a commonly used cloning vector that contains both the ampicillin and tetracycline resistance genes as selectable markers.
It is one of the most popular and commercially used early purpose built plasmid based vector. Inserts cloned in this vector may be characterized easily using this data. Vector pbr322 this artificial vector was constructed by bolivar and rodriguez in 1977. As a starting material for cloning of casein gene sequences. Promoter p1 is artificially created by the ligation of two different dna fragments to create pbr322. The gene for chloramphenicol resistance cm has also been introduced. Thermo scientific pbr322 is one of the most commonly used li cloning vectors. Pbr322 pbr322 low copy cloning vector plasmid vector for molecular cloning. Escherichia coli cells transformed with pbr322 plasmids have a selective advantage in antibiotictreated environments. In order to use antibiotic resistance as a negative as well as a positive selection system, the plasmid vector must carry two different antibiotic resistance genes. This plasmid has a low copy number 20 copies per cell due to the rop gene. Organization of transcriptional signals in plasmids pbr322.
Protocol pbr322 restriction analysis of plasmid dna. Plasmid pbr322 is one of the most widely employed cloning vehicles. 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. Sequence analysis suggests that this transposon is related to transposons tn9 and tn981 but is longer. The vector pbr322 was constructed in order to have a plasmid with a single psti site, located in the ampicillinresistant gene apr, in addition to four unique restriction sites, ecori, hindiii, bamhi and sali. The plasmid pbr322 was one of the fast ek2 mtipose cloning vectors to be designed and constructed. The most widely used of the early purposebuilt vectors is pbr322. Biology 335 lecture notes recdna plasmid vectors i. Some important plasmid vectors pbr322 this was one of the first artificial cloning vectors to be constructed, and is undoubtedly the most widely used cloning vector till now bolivar and rodriguez, 1977a. These fragments can serve as accurate dna size markers from small pieces up to the 4362 base pair length of pbr322. These units are transcribed with various efficiencies, depending upon the individual promoter strengths, which differ in pbr322 up to 10fold. Recombinant dna pancasila pdf pbr322 cloning vehicle messenger rna purification hybridizations. Terms and conditions 20042018 harvard medical school plasmid was created and is maintained by the dfhcc dna resource core at harvard medical schooldfhcc dna resource core at harvard medical school.
Promoter p3 is the natural promoter for the betalactamase gene. The molecule is a doublestranded circle 4,361 base pairs in length 2. The plasmid cloning vector pbr322, shown here, is cleaved with the restriction endonuclease psti. The vector therefore contains features that allow for the convenient insertion or removal of a dna. The plasmid pbr325 is a cloning vector constructed in vitro by the addition of the chloramphenicol resistance gene of an is1flanked transposon to pbr322. Cosmid vector the cosmid vector is a combination of the plasmid vector and the cos site which allows the target dna to be inserted into the.
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. One of the most common plasmids used is the rplasmid or pbr322. Principles of gene manipulation and genomics pdf 7th ed. The amp r gene originally residing on plasmid r1, tet r from r65. The replication origin of pbr322, which directs multiplication of the cloning vector in host cells is originally from pmb1. The main difference between plasmid and vectors is that plasmid is an extrachromosomal element of mainly bacterial cells whereas vector is a vehicle that carries foreign dna molecules into another cell. Conversion of pbr322based plasmids into broadhostrange.1162 1088 538 506 663 1074 421 141 781 1007 451 305 1277 1363 659 1030 96 297 185 772 1446 1404 383 830 533 332 755 1134 376 420 75 1197 498