Plasmids and Vectors
Instructor Supplement to
pGlo Bacterial Transformation
A more detailed look at plasmids
Origin of
Replication
Multiple
Cloning
Site
Promotor
Site
Antibiotic
Resistance
Gene
Cloning into a Plasmid
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
Asilomar Conference
NIH Guidelines
Self Regulation in Science Milestone
Contents
Specified handling and construction processes
Microorganisms containing recombinant DNA were
prohibited outside of the laboratory
Vectors that sexually move to “unsafe” bacteria
was prohibited
Subsequent modifications
1986 expanded to include animals and plants, and
4 biosafety levels
1994 officially relinquished control of GMO plants
in the environment to EPA and APHIS
The First “Safe” Bacterium
Released in 1976 by Roy Curtiss III at
the University of Alabama
E. coli χ1776
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
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
The Cadillac of Cloning Vectors
pBR322
Clone fragment in one
antibiotic gene
Select for other antibiotic
resistance
Screen for presence of
one resistance gene
(selects against
untransformed bacteria)
and loss of resistance to
interrupted antibiotic
resistance gene (selects
for recombinant
molecule)
pBR322
4,361 bp
EcoRI
Tet
R
Amp
R
APstI
BamHI
Screening bacteria by replica plating
Next Major Advance in
Plasmid(ology)
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
Unique sites (usually)
Insert excision facilitated
Restriction endonuclease mapping and Subcloning
made easier
Another Major Advance: Blue-White Screening
•
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
Features of many modern Plasmids
The Major Limitation of Cloning in Plasmids
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)
o
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
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:
/>
Bacteriophage lambda
Protein capsule of
lambda has a tight
constraint on the
amount of DNA
that will fit inside
it (~ 55kb)
By the early
1970’s we knew
that a good
portion of lambda
was not required
“Junk” DNA
COS site: Cohesive
“sticky” ends
Lysis
Lysogeny
Head
Tail
Replication
Circularized
lambda
ori
Not Quite Bacteriophage lambda
Eliminate the
non-essential
parts of lambda
Can now insert
large pieces of
DNA (~ 20 kb)
COS
Lysis
Head
Tail
Replication
ori
Lambda was great:
Larger insert size
Introducing phage DNA into E.coli by phage infection
is much more efficient than transforming E.coli with
plasmid DNA
Have to work with
plaques
But:
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
cos
Tet
R
EcoRI
21.5 kb
ori
Cos site is the only
requirement for
packaging into
phage particle
Cosmids
Other Vectors
BACs (Bacterial artificial chromosomes)
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
YAC (Yeast Artificial Chromosome)
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?
Screen A DNA library:
Genomic
cDNA
Use Polymerase Chain Reaction (PCR) to
clone gene of interest
25
Genomic Library