Transcript Recombinant DNA

Recombinant DNA
Also Known As…
No, we won’t be making the elusive dog-boy
or elephant-crocodile in the lab…So please
stop asking.
WHAT IS “RECOMBINANT” DNA?
• We already know what DNA is – the genetic
blueprint and all – so let’s focus on the
recombinant part of it.
• “Recombinant” refers to the fact that a sample
of DNA has DNA originating from two different
sources or organisms.
• Basically, we are taking a gene for a trait from
one organism and inserting it into the genome of
a second organism to see if it can be adopted and
used.
• If successful, the second organism can produce
the protein that the inserted, foreign gene is
responsible for making.
Step 1: CUT IT OUT
• The first thing we must do is extract the desired
gene (that we wish to insert into something else)
from the source organism.
• We can isolate the DNA from the source and use
a restriction enzyme such as EcoR I to cut out
the gene. We must make sure there are cut sites
close to the ends of the gene but not in the gene
– we don’t want to cut the gene in half or it is
useless.
• As this is being done, a plasmid from a bacterial
cell is being selected, isolated and copied. The
plasmid will also have a recognition site for the
restriction enzyme used for the foreign gene.
Step 2: INSERT INTO VECTOR
• The bacterial plasmid is our vector for the foreign
gene. This means the plasmid is the mode of
delivery for the foreign gene into the host cell.
• Viruses are another vector being considered for
this role in biotechnology.
• We want the foreign gene to use the sticky ends
produced in step 1 to insert itself into the plasmid.
• Ligase is added in order to seal the breaks in the
DNA backbone.
• This will leave use with several outcomes:
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Normal plasmid…no…
Two plasmids attached…nope…
Genes that have attached together…sorry…
A recombinant plasmid – we want this!
Inserting Foreign DNA into a Plasmid
Step 3: Transformation
• Now we run a process called a transformation in
order to get the recombinant plasmids into the
bacterial cells.
• The bacterial host cells are manipulated in order
to make them more permeable to adoption of the
plasmids. This can be done using electroporators,
gene guns or chemicals such as calcium chloride.
• Once the bacterial cell takes up the recombinant
plasmid, it is referred to as being transformed.
• All of the surviving cells are cultured in the
lab and a COPY is made of this plate!!!
• The only thing is you don’t know what plasmid
has been taken up…the recombinant one or the
normal, unaltered plasmid.
Step 4: TEST FOR COMPETENCY
• Now we have to be able to discern between the
following:
• Cell with no plasmid…no…
• Cell with non-recombinant plasmid…nope…
• Cell with recombinant plasmid in it…BINGO!
• The cell with the recombinant plasmid is called a
competent cell. We want this but we have to be
able to pick it our from the other two cell types.
Step 4: TEST FOR COMPETENCY
• To perform this testing process we must first look
at the plasmid in detail…
• A plasmid is “bonus” DNA – non-essential to
bacterial cell function but beneficial in that it
carries resistance genes to potential threats to
the cell.
• We will use these properties in order to identify
our competent cells. How?
• The foreign gene is inserted into one of the
resistance genes in the plasmid and interrupts the
function of that gene – removing the resistance!
Step 4: TEST FOR COMPETENCY
• Look at the plasmid on the
right as an example…
• If we cut this plasmid with
PST I and insert the foreign
gene within the Ampicillin
resistance gene, the cell is
no longer resistant to
ampicillin.
• The tetracycline gene is not
affected in any way so the
cell with the plasmid in
either form is still resistant
to tetracycline.
• We can use this information
to help us identify
competent cells!
Step 4: TEST FOR COMPETENCY
• Keeping our plasmid in mind…
• Along with our three cell types…
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•
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First lets treat the cells with Tetracycline!
Second lets treat the remaining cells with Ampicillin!
Nooooooooooo! We just killed the cells we wanted!
But wait! We made a copy of the bacterial colonies
before the testing!
Step 4: TEST FOR COMPETENCY
• This is what the bacterial plate might look like
during testing…can you tell which colonies were
formed by competent cells?
• Test 1: Tetracycline!
• Test 2: Ampicillin!
• Obviously, colonies
2 & 4 have the
recombinant plasmid
inside of them.
• No go back to the copy
of this plate and grow them!
Step 5: GROW BACTERIA AND CASH!!!
• Once you have grown many copies of the
recombinant bacterial cultures you can harvest
the desired product of the foreign gene and make
lots of money because bacteria work for super
cheap!
FIN