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
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

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 1776
Required

diaminopimelic acid (DAP)

Fragile cell walls (low salt, detergent
sensitive)
Difficult to work with
Slow grower
Poor receptor for transformation

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

The Cadillac of Cloning Vectors

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)

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, pUC18s
polylinker
Sites for 13 REs
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

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

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 ()
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

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
1970s we knew
lambda
that a good
Lysogeny
portion of lambda
was not required
Junk DNA

Not Quite Bacteriophage lambda

Eliminate the
non-essential
parts of lambda
Can now insert
large pieces of
DNA (~ 20 kb)

Lysis
Replication
ori

COS

Head

Tail

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

But:

Have to work with

plaques

Cosmids

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
TetR

21.5 kb

cos
EcoRI
Cos site is the only
requirement for
packaging into
phage particle

Other Vectors

BACs (Bacterial artificial chromosomes)

YAC (Yeast Artificial Chromosome)

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?

Screen A DNA library:

Genomic
cDNA

Use Polymerase Chain Reaction (PCR) to

clone gene of interest

Genomic Library

25

cDNA library

What can you do with a library?

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