plasmids and vectors - Instructor Supplement to pGlo Bacterial Transformation

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