4. The correct answer is d. As carbon dioxide is relea ed, it is removed from the
air in the vial by this precipita60n. Since oxygen is being consumed during
cellular resp iration, the tota l gas volu me in th e vial decreases. This causes
pressure to decrease inside the vial, and water begins to enter the pipette.
Laboratory 6: Molecular Biology
YOU MUST KNOW
• The principles of bacterial t ransformation, including h w plasm ids are
engineered an d taken up by cells.
• Factors that affect transformation fficie ncy.
• Th e fun ction of restr iction enzymes and their role in genetic engineering .
• H ow gel electrophoresis separa tes DNA frag ments.
• How to use a standard curve to determi ne the size of unknown DNA
fr agments.
Overview of the Lab
This labor, tory is actually two di fferent exercises. In the first part of he lab,
Bacterial Transformation, yo u use antibiotic-resi. tance plasmids to trans­
form Escherichia coli. In this part of the lab, a gene for resistance to the antibi­
otic ampicillin is introduced into a strain of Escherichia coli that is killed by
ampicillin. If the susceptible bacteri a incorporate th e foreig n DNA, they will
become ampicillin resistant.
In the second part, Gel Electrophoresis, you separate fragments of DNA
fo r further analysis.
Key Concepts I: Bacterial Transformation
Figure 6.1
• Genetic transformation occms when a host organism takes in foreign DNA
and expresses the foreign gene.
• Bacterial cells have a single m ain chrom osome and circular DNA molecules
called plasmids which carry genetic information. All of the genes required for
ba ic survival an d reproduction are found in the single chromosome.
I Plasmids are circular pieces of DNA that exist outside the main bacterial chro­
mosome and carry their own gen es for specia lized functions includi ng resi ­
tance to specific drugs. In genetic engineering, plasmids are one means used t
introduce foreign genes in to a bacterial cell. To understand how this might
work, consider the plasmid in Figure 6.1.
I Some plasmids have the amp R gene, which confers resistance to the antibiotic
ampicillin. E. coli cells containing this p lasmid, termed "+ampR" celis, can survive
and form colonies on LB agar that has been supplemented with ampicillin.
LABORATORY 6. MOLECULAR BIO LOGY
--------------------------------------------------------
329
• In contrast, cells lacking the ampR plasmid, termed «-ampR" cells, are sensiti\
to the antibiotic, which kills them. An ampicillin-sensitive cell (-ampR) can
transformed to an ampicillin-resistant (+ampR) cell by its uptake of a forei c
plasmid containing the ampR gene.
I Competent cells are cells that are most likely to take up extracellular DNA. Com­
petent cells are in logarithmic growth, and chemical conditions are modified
induce the uptake of DNA. Study Figure 6.2 below to review the lab procedUfi
used to prepare competent cells and get them to take up the ampR plasmids.
eampR plasmids
are added to
experimental
cells only.
~.
E
•
'l~
. .0
~
E)Cel ls are heat­
shocked at 42°C.
Some of the
competent cells
take up the ampR
plasmid and are
transforme d.
O The treated
cell s are spread
on an agar plate
containing
ampicill in.
LB/Ampicil lin
agar
agar
o
Ampicil lin kills the cells that
lack the ampR gene .
Only colonies of E. coli that have been
transformed by the ampR gene will grow.
Figure 6.2
oJ
t
:;;
I
•
I
330
Study Figure 6.3. It shows the expected results in this experiment.
If there is no ampicillin in the agar, E. coli will cover the plate with so many cells
it is called a "lawn" of cells (A + C).
Only transformed cells can grow on agar with ampicillin. Since only some of
the cells exposed to the ampR plasmids will actually take them in, only some
cells will be transformed. Thus, you will see only individual colonies on the
plate (D).
If none of the sensitive E. coli cells have been transformed, nothing will grow on
the agar with ampicillin (B) .
Restriction enzymes or endonucleases are bacterial enzymes that will cut DNA
at specific DNA sequences known as recognition sites. Often the enzymes cut
the DNA so that the ends are single-stranded "sticky ends." A gene of interest
(such as antibiotic resistance) can be introduced into a plasmid.
Here are the general steps used to introduce a gene of interest into bacteria:
1. Both the gene of interest and the plasmid are cut with the same restriction
enzyme, so they have the same sticky ends.
2. DNA ligase is used to anneal and seal the sticky ends.
PART III : THE LABORATORY
No ampic illin
in growth medium
Control 2 No ampR
plasm ids added
Experimental tube 1
ampR plasmid s added
Experimental tu be 2
ampR plasmids added
Ampicillin
in growth medium
No ampici ll in
in growth medium
Amp icillin
in growth medium
Figure 6.3
3. The recipient cells are transformed with the engineered plasmid.
4. Colonies carrying the plasmid are isolated.
I How do we know that transformation has been successful?
1. Use a selection gene, such as for antibiotic resistance. Only those cells that
have incorporated the plasmid will have antibiotic resistance.
2. Use a reporter gene such as GFP (green fluorescent protein). Transformed
cells will glow!
I Transformation efficiency is the number of transformed cells per microgram
of the plasmid. High transformation efficiencies require cells that are in log
phase of growth, suspended in ice-cold calcium chloride, have a rapid heat
shock (this makes the membrane permeable to the plasmid), and plasmids that
are not too large.
Key Concepts II: Restriction Enzyme Cleavage of DNA and Gel Electrophoresis
~
Gel electrophoresis is a procedure that separates molecules on the basis of
their rate of movement through a gel under the influence of an electrical field.
~ The direction of movement is affected by the charge of the molecules, and the
rate of movement is affected by their size and shape, the density of the gel, and
the strength of the electrical field.
I DNA is a negatively charged molecule, so it will move toward the positive pole
of the gel when a current is applied. When DNA has been cut by restriction
enzymes, the different-sized fragments will migrate at different rates. Because
the smallest fragments move the most quickly, they will migrate the farthest
during the time the current is on. Keep in mind that the length of each frag­
ment is measured in number of DNA base pairs.
I In your laboratory you will be given three samples of DNA obtained from a virus,
the bacteriophage lambda. One sample will be uncut DNA, one will be incubated
with the restriction enzyme HindIII, and one will be incubated with EcoRI. You
LABORATORY 6: MOLE CULAR BIOLOG Y
331
will separate the fragments of DNA by electrophoresis, stain the DNA for visual­
ization, and determine the fragment sizes formed in the EcoRI digest.
I The samples are loaded into wells in the gel, and then electricity is applied. Th
DNA fragments will migrate. Remember this!
1. DNA is negatively charged and will migrate toward the positive pole.
2. Smallerfragments ofDNA will migrate faster than larger fragments.
I After electrophoresis, you must stain the DNA for visualization. You submerg
the entire gel in methylene blue, which wilJ bind to the DNA. You then rin
the gel repeatedly with water so that the dye washes off the gel. The DNA wi
appear as blue bands that are easily seen when a light is passed through the gel
I Each fragment of DNA is a particular number of nucleotides, or base pair
long. When researchers want to determine the size of DNA fragments prr.­
duced with particular restriction enzymes, they run the unknown DNA alo ng­
side DNA with known fragment sizes. The known DNA acts as a marker.
your laboratory, the DNA that has been cut with HindIII is the marker; you
will use it to help you determine the fragment sizes in the EcoRI digest.
I Figure 6.4 shows the results of electrophoresis. Semilog paper is used to plo
the results of the HindIlI digest. Since its fragments sizes are known, this is the
standard curve. It can now be used to determine the other fragment sizes from
DNA I and DNA n by interpolation.
100,000
10,000
I\.
Cl
.D
J
1,000
"
r,
~i
,
I
1
-
1000
10
20
30
40
50
Distance (mm)
DNA
Hin~ lIil l
1111 II
I
..
II I I19." 10 .1?P " pq,
DNA II I
I CD I
DNA
Figure 6.4
332
PART III THE LABORATORY
4q
,I~R 160