Selecting Cells with Plasmid Vector
Many cells will not take up plasmid during
transformation
 Cells with plasmid can be identified because
original plasmid contained gene for antibiotic
resistance (ampicillin)
 Use medium with ampicillin – if bacteria grow
then plasmid must be present
 But don’t know if plasmid had DNA insert

Selecting Cells with Plasmid that
Carries DNA Insert
Use beta-galactosidase system to do bluewhite screening
 Host must lack enzyme and plasmid vector
must carry gene for beta-gal
 Restriction site is within beta-gal gene
 Thus, if insert occurred within beta-gal gene,
enzyme not produced
 X-gal = artificial substrate added to medium
that turns blue if enzyme present, otherwise
bacteria are normal white color

Cloning Considerations
 Choosing
a vector
• Plasmids limited to small molecules
• Bacteriophages (phages) are viruses that
infect bacteria
• Phages can carry DNA inserts up to
15,000 nucleotides long
Cloning Considerations
Choosing a host
• Bacteria very good, but limitations
• Bacteria lack ability to modify proteins and limited
size of insert
• Yeast (Saccharomyces cere isiae) excellent for
many applications
• Occasionally necessary to clone genes into
specific animal or plant hosts – more difficult but
possible
Hosts & Vectors

Host systems:
- Bacterium (E.coli)
- Yeast (Saccharomyces cerevisea)
- Insect cells
- Mammalian (Chinese Hamster Ovary cells)

Cloning vectors
- derived from natural replicons
- Capable of replicating and isolation from host.
- Contain a selectable marker to distinguish host cells
containing the vector from amongst those that do not (eg.
antibiotic resistancy or survival under certain growth
conditions.
Types of Vectors




Plasmid DNA
E. coli vectors, extra-chromosomal and circular
Bacteriophages
Phage l – clone large DNA fragments and incorporate
into host genome
Phage M13 – allows cloned DNA to be isolated in
single-stranded form
Cosmids
hybrids of plasmid-bacteriophage l
Artificial chromosomes
- Cloning of very large genomic fragments
- BACs (bacterial artificial chromosomes)
- YACs (yeast artificial chromosomes
Types of Vectors
Types
Plasmid
Lambda phage
Cosmids
BACs
YACs
Inserts
<10000 bp
10-15 kbp
45 kbp
300kbp
1000kbp
Vectors used in different Hosts

Bacteria
E. coli cloning and expression vectors
eg. pGEMT from Promega; pGEX from Invitrogen
pQE from Qiagen

Yeast
yeast episomal plasmids for gene expression
eg. PICHIA expression vectors from Invitrogen

Plants
Agrobacterium tumefaciens Ti plasmid introduce genes
into plants

Eukaryotic cells
Plasmid vectors used for gene expression and functional
studies eg. Viruses – SV 40, baculovirus, retroviruses
• viruses that infect bacteria
• known dsDNA sequence of ~ 50 kb
• linear double-stranded molecule with
single-stranded complementary ends
• cohesive termini (cos region)
http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif
Desirable properties of λ phage:
• can accept large pieces of foreign DNA
• tremendous improvement over the years
• can be reconstituted in vitro
Bacteriophage l
l phage genome linear 48.5 kb
genome.
Each ends consists
of cos (cohesive) sites
– 12 bp cos ends
Cos ends allows
DNA circularization in
the cell
Central region of
genome are nonessential portions and
can be replaced by
foreign DNA (up to
23kb)
Bacteriophage l

Phage particles
injects linear DNA
into the cell

DNA ligate to form
circle

Replicate to form
many new phage
particles which are
released by cell
lysis and cell
death

or DNA intergrate
to host genome by
site-specific
recombination
(lysogenic phase)
Lysis plaques of l phage on
E. coli bacteria.
bacteria lawn
plaques
l bacteriophage
Plaques: the clear areas within the lawn
where lysis and re-infection have prevented
the cells from growing.
M13 phage vectors
1. Replication form (RF, dsDNA) of M13
phage can be purified and manipulated
like a plasmid.
2. Phage particles (ssDNA): DNA can be
isolated in a single-stranded form
•
•
DNA sequencing
Site-directed mutagenesis
Cloning (RF, like plasmid)  transfection
(recombinant DNA)  growth (plating on a
cell lawn)  plaques formation (slow growth)
Hybrid plasmid-M13 vectors
• Small plasmid vectors (pBluescript)
being developed to incorporate M13
functionality
• Contain both the plasmid and M13 origin
of replication
• Normally propagate as true plasmids
• Can be induced to form single-stranded
phage particles by infection of the host cell
with a helper phage.
M13 phage




M13 phage contains a circular 6.7kb ssDNA
Replicate in E. coli cells as double-stranded circles
(replicative form, RF), ~ 100 copies per cell
Cells are not lysed by M13, but grow slowly.
Recombinant M13 phage can produce either
- dsDNA RF can be isolated & manipulated as plasmid
- ssDNA isolated from phage particles in growth medium
( used for DNA sequencing and site-directed
mutagenesis)
M13 phage cloning vectors

M13 RF containing cloned fragment (eg. M13amp18 and 19)
- Transfect into E.coli cells
- plating in a lawn of cells produce plaques
- Plaques consist of slow growth rather than lysis of infected cells
- Blue-white selection using MCS and lacZ

Hybrid plasmid – M13 vectors (eg. pBlueScript)
- eveloped to incorporate M13 functionality
- contain plasmid & M13 origin of replication, minus the genes for full
phage life cycle.
- propagate as true plasmid
- can be induced to form single-stranded phage particles by
infection of the host cell with a helper phage, provides the gene products
required for ss production and packaging
Cosmid vectors
1. Utilizing the properties of the phage l
cos sites in a plasmid vector.
2. A combination of the plasmid vector
and the COS site which allows the
target DNA to be inserted into the l
head.
3. The insert can be 37-52 kb.
Formation of a cosmid clone
Digestion
Ligation
• small circular dsDNA that autonomously replicates
apart from the chromosome of the host cell
• “molecular parasites”
• carry one or more genes some of which confer
resistance to certain antibiotics
• origin of replication (ORI) --- a region of DNA that
allows multiplication of the plasmid within the
host
• plasmid replication: stringent or relaxed
Desirable properties of plasmids:
 small size
 known DNA sequence
 high copy number
 a selectable marker
 a second selectable gene
 large number of unique restriction sites
http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif
• modified plasmids containing cos sequences
• carry an ORI & an antibiotic resistance marker
• can accommodate ~35 to 45 kb of foreign DNA
• can be propagated as plasmids
• can be introduced into host by standard
procedures
• chief technical problems occur when used for
library construction
Still remember
transformation?
JUST A SUMMARY !!!
 Means of inserting foreign DNA into the vector
Ligation of the DNA into the linearized vector
http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/ligation.gif
Requirements for a ligation reaction:
• two or more fragments of DNA (blunt/cohesive)
• buffer containing ATP
• T4 DNA ligase
 Method of placing the in vitro modified DNA
into the host cell
Transformation into the host cell
• bacterial cells take up naked DNA molecules
• cells are made “competent”
• cells treated with ice-cold CaCl2 then heat-shocked
• efficiency of 107 to 108 transformed colonies/μg DNA
• maximum transformation frequency of 10-3
• “electric field-mediated membrane
permeabilization”
• high strength electric field in the
presence of DNA
• protocols differ for various species
• efficiencies of 109 per μg DNA (3 kb)
& 106 (136 kb)
http://bme.pe.u-tokyo.ac.jp/research/ep/img/electroporation.jpg
Electroporation of the DNA into the host cell