Vectors

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Vectors

• Core 218
• Spring 2007
• Cloning Vectors and Cosmids

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What is a Cloning Vehicle or Vector?

• A vector is a DNA molecule in which a foreign
  piece of DNA can be inserted without loss of the
  vectors ability to self replicate.
• A vector may be a plasmid, cosmid, artificial
  yeast chromosome, or virus.

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Essential Features of Plasmids

• Most vectors currently in use carry a replicon
  from pMB1 or Col E1
• Both pMB1 and ColE1 are large plasmids obtained
  from clinical isolates of E. coli
• These maintain 15-20 copies in a cell

FOR MORE INFO...
Hershfield, V., H. W. Boyer, C. Yanofsky, M. A.
Lovett, and D. R. Helinski. 1974. Plasmid ColE1
as a molecular vehicle for cloning and
amplification of DNA. Proc. Natl. Acad. Sci.
713455
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Essential Features of Plasmids

• Plasmids such as pMB1 and ColE1 (multicopy
  plasmids) replicate in a relaxed fashion
• Replication functions are not encoded on the
  plasmid
• Uses functions from the host genes
• DNA polymerases I and III, DNA-dependent RNA
  polymerase, etc.

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Importance of relaxed replication

• Plasmids can function in the absence of ongoing
  protein synthesis absence of host replication
• You can inhibit protein synthesis with
  antibiotics (chloramphenicol, spectinomycin)

FOR MORE INFO...
Staudenbauer, W. L. 1978. Structure and
replication of the colicin E1 plasmid. Curr. Top.
Microbiol. Immunol. 8393.
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Importance of relaxed replication

• Host chromosome replication is prevented
• Plasmids carrying the pMB1 or ColE1 replicon will
  continue to replicate
• Two to three thousand copies per cell
• Why is this important?

FOR MORE INFO...
Clewell, D. B. 1972. Nature of Col E1 plasmid
replication in Escherichia coli in the presence
of chloramphenicol. J. Bacteriol. 110667.
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pMB1 and ColE1 replicons
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pMB1 and ColE1 Replicons

• So what will happen if we alter RNA I or rop?
• Decrease negative regulation of RNA II
• More RNA II available
• More plasmid replication
• Example
• pUC plasmids have a single mutation (G-gtA)
  one nucleotide upstream of the initiation of RNA
  I.
• pUC plasmids have 500-700 copies per cell

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Replicons Also Control Plasmid Compatibility

• Plasmid compatibility the ability of two
  different plasmids to co-exist in the same host
• Plasmids that utilize the same replication system
  cannot co-exist in the same bacterial cell
• Plasmids carrying the same replicon belong to the
  same incompatibility group

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Over 30 Incompatibility Groups are Known
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Plasmid Safety

• Some naturally-occurring plasmids can be
  transferred to hew hosts by conjugation
• Conjugation requires three elements
• A trans-acting mobilization gene (mob)
• A cis-acting element (bom)
• A specific site that is nicked by mob (nic)

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Plasmid Safety

• Some of the older plasmids such as pBR322 are
  missing mob
• Some of the newer vectors such as pUC are missing
  the nic/bom and cannot be mobilized

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In the 1970s and 1980s

• The first cloning vectors such as pSC101 had
  limited functionality
• The next trend was to develop smaller plasmids
• Advantages
• Increased efficiency of transformation
• Easier to restriction map
• Higher copy numbers

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Next Major Advance Polylinkers

• Polylinker is a tandem array of restriction
  endonuclease sites in a very short expanse of DNA
• For example, pUC118s polylinker
• Sites for 13 REs
• Region spans the equivalent of 20 amino acids or
  60 nucleotides

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(No Transcript)
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The Polylinker Advantage

• Unique sites (usually)
• Insert excision facilitated
• Restriction endonuclease mapping and
• Subcloning made easier

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Polylinker from pUC118
EcoR1 SacI KpnI SmaI BamHI XbaI SalI PstI
SphI HindIII
XmaI AccI

HincII
Digest with BamHI
Add BamHI digested target DNA
Ligated rDNA
EcoR1 SacI KpnI SmaI BamHI
XmaI
BamHI XbaI SalI PstI SphI HindIII
AccI
HincII
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Major Advance Blue/White Screening

• E. coli normally produces ?-galactosidase
• Production is under control by the lac operon

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Z beta galactosidase, Y lactose permease. A
thiogalactoside transactylase, lacI repressor,
Pi promoter for the lac repressor, P and O
promoter and operator
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The lac operon with lactose present
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The lac operon when lactose is absent
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Major Advance Blue/White Screening

• Host E. coli strains have a deletion in the
  N-terminus region of the lacZ gene
• These cloning vectors have the P, O and first 58
  amino acids of the lacZ gene (alpha peptide)
• Intact, the vector in an alpha mutant strain (ex.
  JM109) produces beta-galactosidase

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Major Advance Blue/White Screening

• When insert DNA in cloned into the polylinker
  separating the start codon and the alpha peptide
  sequence in the vector, the peptide is not made

• Hence, bacterial colonies containing vector plus
  insert DNA are white

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Major Advance Blue/White Screening

• Inducer ISOPROPYL-ß-D-THIOGALACTOPYRANOSIDE
  (IPTG)
• Colorimetric indicator 5-Bromo-4-chloro-3-indoxyl-
  beta-D-galactopyranoside (X-gal), colonies turn
  blue

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The Major Limitation of Cloning in Plasmids

• Upper limit for clone DNA size is 10 kb
• Requires the preparation of competent host
  cells
• If the E. coli genome contains 4,639,221 base
  pairs, how many plasmid clones are needed to
  clone the entire genome?

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The Major Limitation of Cloning in Plasmids

• Inefficient from generating genomic libraries as
  overlapping regions needed to place in proper
  sequence
• Preference for smaller clones to be transformed
• Lose large genetic regions

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Bacteriophage lambda (?)
http//phage.bocklabs.wisc.edu/Virus.htm In 1971
Alan Campbell showed that the central third of
the genome was not required for lytic growth.
People started to replace it with E. coli DNA
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Lambda genome is approximately 49 kb in
length. Only 30 kb is required for lytic
growth. Thus, one could clone 19 kb of foreign
DNA. Packaging efficiency 78-105 of the lambda
genome.
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Advantages and Disadvantages of lamba cloning

• Larger insert size
• Need to go through lysis to obtain DNA

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What are the Major Features of and Ideal Plasmid
Cloning Vector?

• A replicon containing the origin of replication
• Lack mob site
• Lack nic site
• Lack bom site
• Histochemical differentiation

• Small size
• Able to leap tall buildings in a single bound
• Amplifiable
• Multiple copy number