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How are recombinant plasmids moved through the
membrane into the receiving bacterium.The story
in the high school bio textbook is simply that
restriction enzymes clip the gene of interest,
it is recombined with a ligase, and then this
transformed plasmid is reintroduced into the cell
- but no real explanation. Can you explain this
process?
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Question Date: 2000-01-21 | | Answer 1:
It's amazing how much more complex high school
biology has become just since I graduated in 1990,
we didn't even touch on these topics back in the
80s! I always find that when a textbook is vague
about something, it's usually because the
mechanism is poorly understood (or at least was
when the text was written). This is always a
difficult concept in and of itself to teach
students--that in a biological process, steps 1,
2, 3, 6, 7, and 8 are fully understood, but steps
4 and 5 nobody has a clue about. Anyway, here's
what I know: to make bacteria permeable to DNA you
can use "electroporation", which involves exposing
the cells to brief electric shocks of several
thousand volts. These shocks do not kill the cells
but make them temporarily permeable to DNA, it
makes little holes in their membrane that the DNA
plasmids can pass through. After the shock is
stopped, the holes seal up and the membrane is
unharmed.
There have been many different
techniques developed to transfer DNA into
different cell types too. Plant cells, yeast
cells, even mammalian cells can be made to take up
free DNA in a medium. The uptake mechanisms vary
for each cell type. For example, mammalian cells
will readily take up DNA when the DNA is converted
to a fine precipitate by pre-treatment with
Calcium phosphate. | | Answer 2:
Some strains of bacteria have a natural ability to
take up naked DNA from surrounding media; however,
the common lab strains (mostly E. coli) used in
recombinant DNA technology methods do not have
this property. Therefore, they must be treated a
certain way to make them "competent" to be
transformed with the recombinant DNA plasmid.
(Note-it's the bacterial cell that is
"transformed" by virtue of taking in the
recombinant DNA plasmid). Now, the big question
is: You have the DNA in a test tube along with
some bacterial cells, what is the trick for
getting the DNA in? The most common method for
making the cells "competent" to take up DNA is to
treat them with cold CaCl2 (calcium chloride).
Another, more recently developed method is
electroporation (you literally "zap" the cells
with an electrical shock) while keeping the cells
cold. Both methods tend to make the surface of the
bacterial cell somewhat leaky and less "picky"
about what can cross into the cell, but it is
transient. As you might imagine, the properties of
the buffer in which the cells are bathed during
this time is crucial. The cold temperature
probably does at least two things: (1) It prevents
damage to the cell by protease and nucleases that
might get activated by the various treatments (the
enzymes don't work in the cold); and (2) it
prevents the rapid "repair" of the cell surface,
allowing enough time for the naked DNA (which you
add to the tube of competent cells) to diffuse
into the cells.
| | Answer 3:
I think that you have several things kind of
conglomerated. First off, you have to "get" a
gene or a piece of DNA that you want to amplify.
That piece of DNA is then put in a "vector", a
small, circular piece of DNA that has a high copy
number of replication in bacteria and the ability
to be resistant to some type of antibiotic
(typically ampicillin). How does your piece of
DNA that you want get into the vector?
Restriction enzymes. Your piece of DNA (linear)
and the vector DNA (circular) are both cut with
the SAME restriction enzymes and then put together
in a reaction in the presence of ligase. The
ligase will "ligate" [stick together] the pieces
of DNA so that your DNA is now inserted in the
vector to make a new circular DNA that has the
vector and your DNA insert. Once you "make" that
vector/your DNA hybrid (the recombinant plasmid),
it needs to be put in bacterial cells that will
overexpress it. You do that by using special E.
coli cells that have been treated. Because of the
treatment they go through, they are called
"competent". You mix the DNA and the competent
cells together and then heat-shock the cells.
This will cause little holes in the bacteria, and
the bacteria can then take up the DNA. Then you
let the bacteria "recover" for about an hour, and
then SELECT the bacteria with YOUR plasmid by
using ampicillin in the growth media (that the
cells grow in). The circular piece of DNA will
have the genetic information to make the bacteria
resistant to ampicllin (that would otherwise be
killed by the ampicillin).
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