Plasmids and Vectors
Instructor Supplement to
pGlo Bacterial Transformation
A more detailed look at plasmids
Promotor
Site
Origin of
Replication
Antibiotic
Resistance
Gene
Multiple
Cloning
Site
Cloning into a Plasmid
Asilomar Conference
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People believed that “safe” strains of
bacteria, viruses and vectors could be
made in a few weeks
NIH formed the Recombinant DNA
Advisory Committee (RAC)
It took 1 year (1976) before the first
“safe” (EK2 category) line of E. coli
was released
That year, RAC released a set of
guidelines requiring the use of safe
bacteria
NIH Guidelines
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Self Regulation in Science Milestone
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Contents
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Specified handling and construction processes
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Microorganisms containing recombinant DNA were
prohibited outside of the laboratory
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Vectors that sexually move to “unsafe” bacteria
was prohibited
Subsequent modifications
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1986 expanded to include animals and plants, and
4 biosafety levels
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1994 officially relinquished control of GMO plants
in the environment to EPA and APHIS
The First “Safe” Bacterium
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Released in 1976 by Roy Curtiss III at
the University of Alabama
E. coli 1776
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Required diaminopimelic acid (DAP)
Fragile cell walls (low salt, detergent
sensitive)
Difficult to work with
Slow grower
Poor receptor for transformation
In the 1970’s and 1980’s
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The first cloning vectors such as
pSC101 had limited functionality
The next trend was to develop
smaller plasmids
Advantages
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Increased efficiency of
transformation
Easier to restriction map
Higher copy numbers
The Cadillac of Cloning Vectors
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pBR322
 Clone fragment in one
antibiotic gene
 Select for other antibiotic
resistance
AmpR
 Screen for presence of
one resistance gene
(selects against
untransformed bacteria) APstI
and loss of resistance to
interrupted antibiotic
resistance gene (selects
for recombinant
molecule)
EcoRI
TetR
pBR322
4,361 bp
BamHI
Screening bacteria by replica plating
Next Major Advance in
Plasmid(ology)
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The inclusion of
polylinkers into
plasmid vectors
Polylinker is a tandem
array of restriction
endonuclease sites in a
very short expanse of
DNA
For example, pUC18’s
polylinker
 Sites for 13 RE’s
 Region spans the
equivalent of 20
amino acids or 60
nucleotides
Source: Bio-Rad Laboratories
The Polylinker Advantage
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Unique sites (usually)
Insert excision facilitated
Restriction endonuclease mapping and
Subcloning made easier
Another Major Advance: Blue-White Screening
Features of many modern Plasmids
•Small size
•Origin of replication
•Multiple cloning site (MCS)
•Selectable marker genes
•Some are expression vectors and have sequences
that allow RNA polymerase to transcribe genes
•DNA sequencing primers
The Major Limitation of Cloning in Plasmids
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Upper limit for clone DNA size is 12 kb
Requires the preparation of “competent” host
cells
Inefficient for generating genomic libraries as
overlapping regions needed to place in proper
sequence
Preference for smaller clones to be transformed
If it is an expression vector there are often
limitations regarding eukaryotic protein
expression
Bacteriophage lambda (λ)
A virus that infects
bacteria
o 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
o
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%100% of the
lambda genome.
A complete animation of the lytic cycle:
http://www.blackwellpublishing.com/trun/artwork/Animations/Lambda/lambda.html
Bacteriophage lambda
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Protein capsule of
COS site: Cohesive
lambda has a tight
“sticky” ends
constraint on the
Lysis
Head
amount of DNA
Replication
that will fit inside
ori
it (~ 55kb)
Tail
By the early
Circularized
1970’s we knew
lambda
that a good
Lysogeny
portion of lambda
was not required
“Junk” DNA
Not Quite Bacteriophage lambda
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Eliminate the
non-essential
parts of lambda
Can now insert
large pieces of
DNA (~ 20 kb)
Lysis
COS
Replication
ori
Tail
Head
Lambda was great:
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Larger insert size
Introducing phage DNA into E.coli by phage infection
is much more efficient than transforming E.coli with
plasmid DNA
But:
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Have to work with
plaques
Cosmids
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Hybrid vectors: plasmids that
contain bacteriophage lambda
cos sites
DNA (~ 33-48 kb) cloned into
restriction site, the cosmid
packaged into viral particles
and these phages used to infect
E.coli
Cosmid can replicate in
bacterial cell, so infected cells
grow into normal colonies
Insert DNA limited by the
amount of DNA that can fit into
phage capsule
Somewhat unstable, difficult to
maintain
ori
21.5 kb
TetR
cos
EcoRI
Cos site is the only
requirement for
packaging into
phage particle
Other Vectors
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BACs (Bacterial artificial chromosomes)
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YAC (Yeast Artificial Chromosome)
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Large low copy number plasmids (have ori and
selectable marker)
Can be electroporated into E. coli
Useful for sequencing genomes, because insert size
100 - 300kb
Can be grown in E.coli and Yeast
Miniature chromosome (contains ori, selectable
markers, two telomeres, and a centromere
Can accept 200 kb -1000 kb; useful for sequencing
Ti plasmids; to introduce genes into plants
Expression vectors
How do you identify and clone a gene
of interest?
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Screen A DNA library:
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Genomic
cDNA
Use Polymerase Chain Reaction (PCR) to
clone gene of interest
Genomic Library
25
cDNA library
What can you do with a library?
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Can be used to complement a mutant (this is
more common for research in bacteria).
Can use it in a colony hybridization.
Screening libraries
by colony hybridization
Polymerase Chain Reaction
(PCR)
Agarose gel electrophoresis
Restriction Mapping