Transcript lect 6- Types ofCloning Vectors

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
• 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 Nterminal fragment of beta-galactosidase .
COSMIDS
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 pLFR5(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
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)
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.
Yeast Artificial Chromosomes
(YACS )
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