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Trends in Biotechnology
110512 – Ch 3c Vectors
and other ways of
transformation
To selectively kill cells and select for successfully
transformed bacterial cells.
Exact copies of the original bacterial colonies are made
by replica plating from the original master plate.
A sterile cloth pad is placed on the plate, so cells stick
to it.
The pad is pressed onto other plates, creating exact
replicas of the original plate.
Antibiotics in the media in the new plates will kill any
bacteria that do not have the recombinant plasmid
inside of them.
The original plate is then examined to see which
bacterial colonies were successfully transformed.
Fig. 3.7
Selection for
recombinant
cells after
transformation.
Replica plating
Question:
A gene is put into
pBR322 plasmids
using Sal 1.
Which of these
colonies have the
new gene?
pBR322 was made so we could select
colonies that lost antibiotic
resistance.
But other vectors can be made which
show loss of color.
The vectors pUC18 and pUC19 (often
called pUC18/pUC19 together) have a
lacZ’ gene. They can make part of the
-galactosidase protein.
α-complementation
The vectors pUC18 and pUC19 can make
part of the -galactosidase protein (the
first 146 amino acids).
These vectors are put into a bacterium
with the last part of the galactosidase protein.
Together they can make a complete galactosidase protein.
Together they can make a complete galactosidase protein.
This is called α-complementation.
When the -galactosidase enzyme is
made it can hydrolyze lactose into
glucose and galactose.
The enzyme can also change a chemical
called X-gal. This chemical will
become blue.
Fig. 3.8 Restriction map of pUC18/pUC19 showing the ampicillin resistance gene,
the origin of replication, and the multiple cloning site within the lacZ’ gene.
Fig. 3.9 The selection of
bacterial colonies
containing a recombinant
vector (pCU vector +
DNA insert) by the
selection of white
colonies.
Bacteriophage can be engineered into a cloning vector.
They kill bacteria by two pathways:
Lytic pathway (Figure 3.10):
Bacteriophage DNA inserted into the bacteria, phage DNA and
proteins are made, assembled, and burst out of the bacteria,
causing the bacteria to burst (called “lysis”).
Recombinant phage DNA can be inserted into bacteria on a
bacterial plate. The bacteria die, forming clear areas called
“plaques” where the recombinant DNA can be isolated.
Lysogenic pathway (Figure 3.11):
The bacteriophage genome is integrated into the bacterial DNA,
replicating along with the bacterial cell genome.
Engineered to only work when recombinant DNA and
viral particles are packaged in a test tube and
placed on bacterial plates to infect bacteria.
Usually hold DNA up to twenty kilobases (kb) in length.
Recombinant phage DNA can
be inserted into bacteria on a
bacterial plate. The bacteria die,
forming clear areas called
“plaques” where the
recombinant DNA can be
isolated.
Fig. 3.10 The lytic life cycle of a bacteriophage.
Fig. 3.11 The
cloning of
human DNA
restriction
fragments (●
and ▲) into a
bacteriophage
vector.
Cosmids.
A small plasmid that contains the cos
(cohesive termini) sites from phage DNA,
a plasmid origin of replication, and genes
for antibiotic resistance.
Packaged into a bacteriophage protein coat,
but the plasmid replicates like a plasmid
instead of phage DNA once it is in the
bacteria.
Can hold pieces of DNA between thirtyfive and forty-five kilobases.
Yeast Artificial Chromosomes (YACs).
Useful for eukaryotic molecular studies, and
contains the following components:
A centromere allows the chromosome to be
transferred to the daughter cells during cell
division.
A telomere at the end of the yeast chromosome
ensures that the end is correctly replicated and
protects against degradation.
An autonomously replicating sequence (ARS)
that consists of specific DNA sequences that
allows the molecule to replicate.
A marker gene that provides a way to detect an
inserted DNA fragment.
Useful to clone large DNA fragments, between
200 and 1500 kilobases.
Example of a Yeast Artificial Chromosome (YAC).
Bacterial Artificial Chromosomes (BACs).
Synthetic vectors that have been used to clone
very large fragments of eukaryotic
chromosomes, between 100 to 300
kilobases.
Used in the analysis of large portions of
complex genomes, whole genes, and
constructing maps of genomes.
Created using a small plasmid, called the F
(fertility) factor, along with cloning sites and
selectable markers.
The F factor allows the vector to carry larger
pieces of DNA, up to 25% of the size of the
bacterial chromosome.
oriS, repE – F for plasmid
replication and
regulation of copy
number.
parA and parB for
partitioning F plasmid
DNA to daughter cells
during division and
ensures stable
maintenance of the
BAC.
A selectable marker for
antibiotic resistance;
some BACs also have
lacZ at the cloning
site for blue/white
selection.
T7 & Sp6 phage
promoters for
transcription of
inserted genes.
Example of a Bacterial Artificial Chromosome (BAC).
Plant Cloning Vectors.
Used for purposes such as resistance to
disease, pests, and herbicides;
improving crop quality and yield;
improving nutritional quality; and
increasing the life of foods.
Most commonly used plant vectors are
the tobacco mosaic virus and the Ti
plasmid from the soil bacterium
Agrobacterium tumefaciens.
Tobacco mosaic
virus (TMV) is
a positivesense single
stranded RNA
virus that
infects plants.
The infection
causes
patterns
(mottling and
discoloration)
on the leaves.
TMV was the
first virus to be
discovered.
Agrobacterium tumefaciens and the Ti plasmid:
Agrobacterium tumefaciens causes tumor in plants,
caused by T-DNA (transferred DNA), located in the
Ti plasmid, and contains eight genes that integrate
into the plant genome.
Engineered Ti plasmids lack the tumor-causing genes,
but have the genes required to integrate the DNA of
interest into the plant genome.
The plasmid is inserted into a plant embryo either by
soaking seeds with recombinant A. tumefaciens
bacteria, or by inserting the Ti plasmid into cells,
which will give rise to the entire plant.
Selectable marker genes allow selection of only the
plant cells with the plasmid DNA.
Fig. 3.12 The
use of
Agrobacterium
tumefaciens to
transfer DNA
into plants.
Video: Ti plasmid
http://highered.mcgrawhill.com/sites/0072437316/student_view
0/chapter16/animations.html#
Mammalian Cell Vectors. There are several:
Simian virus 40 (SV40) — a small DNA tumor virus,
could only hold a small piece of DNA and caused
only transient (temporary) expression of the
inserted DNA.
Retrovirus — a single-stranded RNA virus that contains
a gene for the enzyme reverse transcriptase to
create double-stranded DNA from RNA template,
so that the DNA can integrate into the host cell’s
genome. It needs to infect actively dividing cells.
Adenovirus — a double-stranded DNA virus that can
infect many types of host cells with high efficiency,
with a low chance for causing disease. It does not
have to infect actively dividing cells.
Mammalian cells are used because
bacteria are not able to produce
complex eukaryotic proteins that are
modified by processes such as
glycosylation, or if the mRNA needs to
be processed after transcription.
Cell Transformation.
Many different cells can have DNA
inserted into them, “transformation.”
Some bacteria are naturally competent to
take in new DNA.
Electroporation
If a cell has a cell wall, such as plants,
algae, or fungi, enzymes break down the
walls. A short electrical pulse opens up
pores in the cell membrane, allowing
DNA to enter in what is called
“electroporation.” The cell wall reforms
and cell division starts.
Gene Guns
Plant cells can also be transformed by
biolistics. Small gold or tungsten
particles covered in DNA are shot into
cells or tissue at high velocity. Cell walls
do not have to be removed.
Fig. 3.13 Particle gun used for transforming cells by biolistics.
An animation of a gene gun can be found
at:
http://www.hort.purdue.edu/hort/course
s/HORT250/animations/Gene%20Gun%
20Animation/Genegun1.html
Animal cells can be transformed by
electroporation or DNA precipitation with
a calcium phosphate solution. Viruses
can also be used.
Microinjection of DNA in the cell
nucleus, such as an animal egg, is
needed to introduce DNA into an
entire animal. The DNA integrates
into the animal chromosomes, the
egg is implanted, and the animal is
born with the desired traits.
Fig. 3.14 Microinjection of DNA into pronucleus of a fertilized animal egg.