Transcript Plasmids and Plasmid Maps

Plasmids and Plasmid Maps
BEGINS
Mr Badbacte Vs Mr Goodbacter.
HI there boys and girls.
I’m Gumby and I will
be teaching you all
about this thing called
the plasmid. We use it
a lot in biotechnology
so it is really
important.
The reason the plasmids are so
important is that they are used by
bacteria in a type of exchange of
genetic information. Its a little like
sex for bacteria –but its called
conjugation. The method is also
called HORIZONTAL GENE TRANSFER.
Hi there partner in crime.
What’s goin’ on here?
Hey when can my
wife come for a
conjugal visit?
You can notice in this bacterial cell that
the plasmids are in blue and the bacterial
genome is in red. The bacterial genome
is circular and it is a lot larger than the
plasmid. It is needed by the bacteria to
perform all the functions necessary for it
to survive. The plasmids are like a little
bonus. They can transfer little pieces of
information like antibiotic resistance for
example. Because of the smaller size of
plasmids, they are easier to move into
bacterial cells
This is what it’s all
about. You see the
plasmid is used as a
vector (method of
transport) for
cloned genes. You
can see this plasmid
has been cut and a
foreign gene
inserted in. The
bacteria with this
plasmid are now the
clones and they can
manufacture a gene
product.
Hey kids. Say hello to my good friend Mr.
Goodbacter. He is a transformed bacteria by
genetic engineering. You can see the little
plasmid in his chin. Say hello!
You lousy traitor.
You’re not supposed
to be helping
humans. You should
be giving them
infections and stuff.
Thanks for the welcome. You know, I’m
really excited about producing great
products to help humanity. So far I
have clones that produce valuable
hormonal products like insulin and
growth hormone. Scientists are
working on many more as we speak.
Say, who’s your friend Mr. Goodbacter?
Hey Goodbacter.
What did these
“scientists” ever
do for you?
His name is Mr. Badbacter and he
tries really hard to give all us
bacteria a bad reputation. He
thinks that we bacteria should all
cause disease, but I know that the
vast majority of bacteria do not
do that. Most bacteria are helpful
for the environment specifically
decompsition. Did you know that
some bacteria can even help clean
up oil spills? I’m helpful in a very
new scientific way, thanks to our
brainy scientists.
So these plasmids offer a lot of
possibilities for the bacteria to produce
things we never thought possible.
Scientists use us because we are easy to
grow and we multiply rapidly . This
makes it easy for them to experiment
and determine if a plasmid gets into a
bacteria. For instance, if they are not
sure that the bacteria obtained a
particular plasmid, they can check by
placing us all on an antibiotic plate. Only
those with the plasmid will survive
because the plasmid transfers antibiotic
resistance.
Plasmids can be cut open and new genes can be
inserted. In order to do this scientists are
interested in where the cuts are located on the
plasmid. It is important, for instance, that when
you use a restriction enzyme (ex EcoRI) it does
not cut through something important already
on the plasmid. For instance, you would not
want the EcoRI to cut in the middle of the
antibiotic resistance gene, because that would
ruin the bacteria’s ability to produce the
resistance to the antibiotic. In order to figure
out where all these genes are and where we
will cut with a specific restriction enzyme, we
need something called a PLASMID MAP
To start to understand how to map a plasmid,
lets take an example. Lets say that a particular
plasmid is only cut once by the restriction
enzyme EcoRI=
scissors
Since the plasmid is a circle, if it has
been cut once, it forms one length of
DNA. Run it on the get to see.
4500bp
4000bp
3500bp
3000bp
This all happens on a petri dish, so
we cannot see it. We need to use
electrophoresis to visualize what has
happened.
2500bp
2000bp
1500bp
500bp
Gel electrophoresis
Sample
is
4000bp
long
To start to understand how to map a plasmid,
lets take an example. Lets say that a particular
plasmid is only cut once by the restriction
enzyme EcoRI=
scissors
Since the plasmid is a circle, if it has
been cut once, it forms one length of
DNA. Run it on the get to see.
Plasmid A
4000 bp
total length
Sample
is
4000bp
long
4500bp
4000bp
3500bp
3000bp
This all happens on a petri dish, so
we cannot see it. We need to use
electrophoresis to visualize what has
happened.
2500bp
2000bp
1500bp
500bp
Gel electrophoresis
So in this instance the plasmid is 4000 bp (nucleotide
base pairs) long. If we try to cut the plasmid with
another restriction endonuclease it will not cut in the
same spot. We will represent this other restriction
enzyme with a different pair of scissors.
Hind III
Plasmid A
4000 bp
total length
Note how the number of
base pairs remains the
same 3000 + 1000 = 4000
From the gel it is
apparent that
there are two
pieces of DNA and
thus two cuts of
the plasmid.
Comparing the
lines to the
reference sample,
one length is
1000bp, the other
3000bp.
This time I’ll let you figure out the number of
cuts and the size of the plasmid. We will use
a new plasmid, Plasmid B, this time.
EcoRI
What can you tell from the gel?
Plasmid B
2500 bp
total
There are two cuts on this
plasmid with restriction enzyme
EcoRI. The two DNA lengths
are 1000 bp and 1500 bp. This
means that the total length of
the plasmid is 2500 bp. A
diagram is provided above.
This time we’ll take a little short cut. Instead of
having you look at the gel, we will just get the
information off a chart.
EcoRI
EcoRi (base pairs)
1800
Plasmid C
1800 bp
total
Results of
restriction
enzyme
digestion of
plasmid C
Notice these
cut at
different
locations
BamHI
Bam HIi (base pairs)
1200
600
Plasmid C
1800 bp
total
Now for the next one we will do a double
digest utilizing two different restriction
endonucleases . EcoRI and BamHI We will
still use plasmid C. Hold on tight!
EcoRI
BamHI
EcoRI +
BamHI
1800 bp
1200 bp
200 bp
600 bp
600 bp
1000 bp
These
add
together
to make
the 1800
total
Determine
which Bam is
spliced by the
EcoRI. Here
1200 is cut to
200 and 1000
in the double
digest
This is a double digest.
The plasmid is placed in a
container with both of
the restriction enzymes
Position the EcoRI
1800/0 start
Plasmid C
1800 bp
total
Position the 1200 Bam so that
the EcoRI cuts it into two pieces
, one 1000bp(dark green) and
the other 200bp(light green)
The reverse orientation is also
correct in this plasmid map
This is the
direction
of the
number
system.
You only have
to choose one.
They are both
correct
Finally after
colour coding
the splices we
make the
final map
1800/0 bp EcoRi
200bp
BamHI
800 bp
BamHI
1800/0 bp EcoRi
1600bp
BamHI
1000bp
BamHI
You will get some experience working on these
plasmid maps from the Plasmid map worksheets you
are to complete. When completing your plasmid map
activity, you are to include one working diagram with
the colour code and base pair numbers on it. You also
will include your final plasmid map showing
restriction endonuclease cuts in the appropriate
locations.
1800/0 bp EcoRi
200bp
BamHI
800 bp
BamHI
The worksheets have been made a little easier for
mapping by utilizing a plasmid of 1200 bp length. This
can be compared to a clock to make interpretation
easier.
EcoRI always at top1200/0
EcoRI
BamHI
EcoRI +
BamHI
1200 bp
800 bp
300 bp
400 bp
400 bp
500 bp
300 bp BamHI
700 bp BamHI
Here is an example of a real plasmid map.
Gee, Mr.
Goodbacter. It
sure is
complicated.
It has to be. A lot of these plasmids have
control sequences to control the inserted
genome . Antibiotic resistant genes also have
been placed already to make the plasmid easy
to identify.
Wow. That must have
taken a lot of work to
design that plasmid
map. It looks like they
have inserted quite a
few foreign genes into
it alreadyl
And here’s one last example showing the more
complicated plasmid maps and the associated gel
Biotech sure is
interesting.
PLASMID AND PLASMID MAPS
END
I’m not licked yet. Maybe I can convince some of these
transformed bacteria to go to the Dark Side. You know,
with the power of genetic manipulation, maybe they
could be even more dangerous.