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CLONING VECTORS. Shumaila Azam. IMPORTANT CLONING VECTORS. 1. pBR322. pBR322 DNA is a commonly used plasmid cloning vector. pBR322 DNA is isolated from E.coli by ion exchange chromatography. The molecule is a double-stranded circle, 4361 base pairs in length. STRUCTURE OF pBR322.
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CLONING VECTORS Shumaila Azam
1. pBR322 • pBR322 DNA is a commonly used plasmid cloning vector. • pBR322 DNA is isolated from E.coli by ion exchange chromatography. • The molecule is a double-stranded circle, 4361 base pairs in length.
STRUCTURE OF pBR322 • pBR322 is 4361 bp in length and contains: (1) the replicon rep responsible for the replication of plasmid (source - plasmid pMB1) (2) rop gene coding for the Rop protein, which promotes conversion of the unstable RNA I - RNA II complex to a stable complex and serves to decrease copy number (3)bla gene, coding for beta-lactamase that confers resistance to ampicillin (4) tet gene, encoding tetracycline resistance protein.
STRUCTURE OF pBR322 • The exact position of genetic elements is shown on the map • The bla gene nucleotides 4153-4085 (complementary strand) code for a signal peptide. • The indicated rep region is sufficient to promote replication. DNA replication initiates at position 2533 and proceeds in the direction indicated.
STRUCTURE OF pBR322 • The map shows enzymes that cut pBR322 DNA once.
pBR322 • The first really useful plasmid for genetic engineering, pBR322, was pieced together by Francisco Bolivar, and others in Herbert Boyer's laboratory in the 1970s. • (The "B" stands for Bolivar and the "R" for Rodriguez, another scientist in Boyer's laboratory). • What makes pBR322 useful is that it contains an ampicillin resistance gene and a tetracycline resistance gene. In addition it has a relaxed origin of replication (indicated by the green arrow shown at the bottom of the illustration) and accumulates to high numbers in E. coli.
2. pUC19 • pUC19 DNA is a commonly used plasmid cloning vector in E.coli. • Isolated from E.coli by ion exchange chromatography. • The molecule is a double-stranded circle, 2686 base pairs in length.
STRUCTURE OF pUC19 • pUC19 plasmids contain: (1) the pMB1 replicon rep responsible for the replication of plasmid. (2) bla gene, coding for beta-lactamase that confers resistance to ampicillin .It differs from that of pBR322 by two point mutations (3) region of E.coli operon lac containing CAP protein binding site, promoter Plac, lac repressor binding site and 5'-terminal part of the lacZ gene encoding the N-terminal fragment of beta-galactosidase .
COSMIDS • Cloning vectors called cosmids can carry 40 kb of cloned and can be maintained as plasmids in E.coli. • Cosmids combine the properties of plasmids and bacteriophage lambda vectors.
COSMIDS • For.example: The commonly used cosmid pLFR-5(approx 6 kb) has two cos sites(cos ends)from bacteriophage lambda separeted by ScaI restriction endonuclease site, a multiple cloning sequence with six unique sites, a multiple cloning sequence with six unique sites, an origin of DNA replication, and a tetracycline resistence gene. • This cosmid can carry about 40 kb of cloned DNA.
PHASMIDS • The phasmids are prepared artificially, by combining features of phages with plasmids, as the name suggests. • The commonly used phasmid in molecular biology is pBluescript 11 Ks. It is derived from pUC19 and is 2961 bp long.
Yeast Artificial Chromosomes (YACS ) • Replicates as linear chromosome in yeast. • Can incorporate 100 kb - >2 Mb of foreign DNA. • Vector contains: • Bacterial ori and ampr. • Yeast centromere and ARS. • Ciliate telomere. • Yeast selectable marker.
