Recombinant Plasmids
What is a plasmid?
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A plasmid is a circular DNA molecule separate from
the much larger bacterial chromosome.
Every plasmid has an origin of replication, required
for its replication within the cell.
In nature, bacteria can transfer DNA
in three ways
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Unlike the processes in animals and plants,
bacterial transfer does not create new
individual cells, but creates new genes by
moving genes from one cell to another.
There are three ways in which gene transfer
can take place:
 Transformation
 Transduction
 Conjugation
Recombination
in nature
Transformation

This is the process of the taking up of DNA from
the fluid surrounding a cell.
Transduction
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The transfer of bacterial genes by a phage. In this process, a fragment of
DNA belonging to the phages’ previous host cell has been accidentally
packaged instead of the phages’ regular DNA. When the phage infects a
new cell, the “accidental” DNA is injected into the new cell.
Conjugation
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The union of cells and the DNA
transfer between them. The “male”
cell has sex pili, and one of them is
attached to the “female” recipient
cell. The outside layers of the cell
have fused, and a cytoplasmic
“bridge” has formed in between
them.
To transfer DNA: The donor cells
DNA passes through this mating
bridge into the recipient cell. The
donor cell replicates its DNA as it
transfers so that the donor cell does
not end up lacking any genes. This
DNA replication process is a special
one that allows one copy to peel off
and transfer into the recipient cell.
The “F” Factor as a piece of DNA

The F. Factor (meaning
fertility factor) allows
bacterial cells (such as a male
E.Coli cells) to carry out
conjugation. It is a specific
piece of DNA that carries
genes for making sex pili and
other things needed to carry
out conjugation. It also
contains an origin of
replication; where DNA
replication can begin.
How the F. Factor behaves during
conjugation
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The F. factor is integrated into the
male bacterium's’ chromosome.
When this cell conjugates with a
recipient cell, the male donor cell
begins replication at the Origin of F
replication.
The growing copy of chromosome
replicating is then peeled off from
the original strand and heads into
the recipient cell through the
cytoplasmic bridge.
How the F. Factor behaves during
conjugation (cont’d..)
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Once the transferred donor genes
are inside the recipient cell, they can
recombine with the corresponding
part of the recipient chromosome by
crossing over.
If crossing over occurs, the recipient
may be genetically changed but it
will usually still be female because
the two cells break apart before the
rest of the F. Factor transfers.
F. Factor as a plasmid
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The F. Factor can also exist as a
plasmid.
Some plasmids, including the F. Factor
plasmid, can bring about conjugation
and replicate its entire plasmid and
move into another cell.
This occurs in the same way a
chromosome would transfer, (replicate,
peel away, cross bridge into other cell)
except when the plasmid enters the
recipient cell, the recipient becomes
“male”. When a transferable plasmid
carries more genes than those
needed for replication and
conjugation, it is acting as a vector.
Different types of plasmids
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E.Coli and other different types of bacteria have many different
types of plasmids.
Some plasmids can affect the survival of the cell.
R. plasmids (a class of plasmids) pose seriously problems for
human medicine. These transferable plasmids are resistant
(hence the R) to antibiotics that would normally kill them. From
the use of antibiotics, bacteria can be killed when it lacks the R.
plasmid, while those with an R. plasmid tend to multiply. As a
result, an increasing number of bacteria that cause human
diseases, like food poisoning and gonorrhea are becoming more
resistant to antibiotics.
However, R. plasmids can be useful vectors for genetic
engineering.
Plasmids are used to customize
bacteria
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Page 234
 1) Plasmid is isolated from bacterium
 2) DNA carrying gene of interest is taken from another cell
 3) A piece of that DNA in inserted into the plasmid, making
a recombinant plasmid
 4) A bacterial cell takes up the plasmid by transformation
 5) What is now a genetically engineered recombinant
bacteria is cloned to make many copies of the gene.
Recombinant
Plasmids
made by DNA
technology
Cut and Pasting DNA
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This means cutting a gene out of one DNA molecule
and putting it into another (“cutting” the gene of
interest out of a chromosome and pasting into a
bacterial plasmid)
Cutting tool in a test tube are restriction enzymes

In nature, restriction enzymes this enzyme protects
bacterial DNA from intruding. It works by chopping up the
foreign piece of DNA, a process named restriction because
it prevents foreign DNA from surviving in the cell.
This cutting tool recognizes short nucleotide sequences in
DNA molecules and cuts at specific points within these
recognition sequences.
Cutting DNA
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Pg. 235
1) A piece of DNA containing one recognition sequence for a particular
restriction enzyme. (The place where the DNA is cut is called a restriction site)
2) The cuts yield two double-stranded DNA fragments with single-stranded
ends, called “sticky ends”.
Cutting DNA (cont’d)
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2 cont’d) Sticky ends are used to join DNA restriction fragments from
different sources. They are able to base pair with any DNA molecule
with containing the complimenting sticky end.
3) A piece of DNA from another source (the grey strand). Notice that the
grey strand has a single stranded end identical to the base sequence of
the sticky ends on the blue DNA.

Why do these have the same base sequences?
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It has ends with this particular sequence because it was cut from a larger molecule
by the same restriction enzyme used to cut the blue DNA.
4) The complimentary ends on the blue and grey fragments stick
together by base pairing and hydrogen bonds.
Pasting DNA
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The combination of the grey and blue DNA fragments is only
temporary, because only a few hydrogen bonds hold it together. If it is
wanted to be permanent, you can “paste” then together by using DNA
ligase.

DNA ligase: The cell usually uses this in DNA replication. It
catalyzes the formation of covalent bonds between adjacent
nucleotides, sealing the “bubbles” in the DNA strands.
Applications of genetically
engineered bacteria
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Recombinant Plasmids are extremely useful in society. It is because it is now
possible to create unlimited amounts of whatever needed. These new
cultured cells are used to manufacture
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Protein used to dissolve blood clots in heart attack therapy (tissue plasminogen
activator)
Insulin
Hepatitis B surface antigen, to vaccinate against the Hep B virus
Granulocyte-macrophage colony-stimulating factor (GM-CSF) for helping stimulate the
bone marrow after a bone marrow transplant
Gene for pest resistance that is inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
To make recombinant DNA useful it must be made in large
amounts.
Cloning Genes in Recombinant
Plasmids
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In order for recombinant DNA to be useful it must be made in vast amounts.
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Ex. A scientist finds a human gene that codes for a valuable product – the
biologist to make a system where there is large amounts of the gene produced so
it can be sold on a large scale
Step 1 : Isolate 2 different DNA : bacterial plasmid that serves as vector, and
human DNA of interest
Step 2: Treats both DNA with same restriction enzyme. Plasmid is cut in one
place, DNA of interest is cut in many fragments – one including the gene
of interest.
Step 3: Gene of interest is mixed with cut plasmid. Sticky ends attach.
Step 4: DNA ligase joins two DNA molecules with covalent bonds. Result :
Recombinant DNA plasmid containing gene of interest.
Step 5: Plasmids is added to a bacterium. In right conditions, bacteria will
take up plasmid by transformation
Step 6: Gene Cloning – Bacteria reproduces
Bibliography
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http://users.rcn.com/jkimball.ma.ultranet/Biolo
gyPages/R/RecombinantDNA.html#kanamyci
n
Biology : Concepts and Connections