Download BIO105 Principles of Biology Transformation

BIO105
Principles of Biology
Transformation
Introduction:
The transfer of genetic information between bacteria has been occurring for billions of years.
Bacterial transformation was observed in 1920’s by Frederick Griffith, who identified the
transformation of one strain of avirulent, rough-coated Streptococcus pneumoniae to another strain
that was smooth-coated and virulent (=causing disease). The transforming factor was later
investigated in depth by Avery and his co-workers in the 1940’s at what is now the Rockefeller
University. Extensive experiments and analysis suggested to those investigators that DNA was the
heritable material, but their conclusions were not widely recognized or acknowledged
Chromosomal DNA from one bacterial strain can be transferred to another, unrelated species of
bacterium, but this occurs only infrequently. Linear fragments of DNA (such as chromosomal
fragments) are easily broken down outside a living cell, and many agents in the medium
surrounding dying cells can modify the structure of DNA. More commonly, plasmid DNA is
involved in natural transformations. Plasmids are small circular molecules of DNA that exist in the
cell separately from the bacterial chromosome. There can be different kinds of plasmids within the
same cell, as well as multiple copies of a single plasmid. Plasmids are often found associated with
bacterial cells and are thought to be responsible for considerable diversity and versatility in bacterial
metabolism. Plasmids are also used in biotechnology applications to accomplish the rapid transfer
of genetic information between bacterial cells.
In this lab we are going to carry out a transformation using pLUX. This plasmid is a recombinant
plasmid containing the genes from Vibrio fischeri that are involved in bioluminescence. Various
Vibrio species are found in marine habitats, both free living and in symbiotic associations. Vibrio
fischeri in particular is known to exist in pure cultures within the light emitting organs of squid and
several kinds of fish. Bioluminescence has been known for a long time, with an early description of
it by Aristotle, who observed that rotten fish glows in the dark! Almost certainly this was an
instance of bacterial bioluminescence on the surface of the rotting fish. Biology departments often
get calls from members of the general public reporting these same instances of “glowing” flesh on
old fish or other materials of marine origin. A variety of organisms have capacities for
bioluminescence and many possible explanations of its significance have been advanced.
Bioluminescence may be part of an escape response (jellyfish), involved in peculiar pathways of
oxidation-reduction (bacteria, dinoflagellates), or as species recognition signals (squid, fish).
Anyone who has seen bioluminescence will testify to its fascination!
In bacteria, genes for a specific process are often found clustered into an operon. An operon is an
arrangement of several genes such that transcription of all the genes can occur from a single
promoter; because the cell is able to control production of messenger RNA from all these genes at
the same time, this is an effective way to co-regulate the production of many different proteins. The
LUX operon of V. fischeri is a set of genes responsible for expression of proteins involved in
bioluminescence. Using restriction endonucleases and DNA ligase enzymes, the Vibrio LUX
operon has been inserted into the pBR322 plasmid. The pBR322 plasmid also contains a gene for
resistance to the antibiotic Ampicillin.
In this lab, you will attempt to accomplish the
transformation of non-bioluminescent Eschericia coli by getting the E. coli cells to take up the new
plasmid (plasmid pLUX).
After transforming the E. coli cells, we will spread them onto a nutrient agar plate that contains
ampicillin. Ampicillin is a penicillin-like antibiotic that inhibits bacterial growth by interfering with
the ability to synthesize new bacterial cell walls. There is a gene on pLUX that encodes a protein
which confers ampicillin resistance; because the original E. coli cells are not resistant to ampicillin,
only the cells that have taken up pLUX will be able to grow and form colonies on the plate. The
ampicillin-resistance gene encodes a protein that is able to cleave ampicillin. It is neceassary to use
this 'selectable marker gene' because transformation is a rare event, and only about 1 in every 10
million cells will be transformed using this procedure. It would be very difficult to separate out
those few transformed cells from the rest of the cells if we didn't use a selectable marker gene.
Cloning DNA in Plasmids.
DNA of any origin (including human) can be inserted into a plasmid (a small, selfreplicating circular molecule of DNA) in a process called cloning. The resulting
recombinant plasmid is introduced into rapidly reproducing bacterial cells via
transformation protocols such as the one described in this exercise. The newly inserted
segment will be replicated along with the rest of the plasmid, and proteins encoded by
genes on the inserted DNA can be made in the bacterial cells. In our experiment, the
genes necessary for the production of luciferase are on the inserted region, and will be
expressed by the transformed E. coli, allowing them to glow in the dark.
Protocol: Transformation.
1. Obtain two tubes of competent cells from the refrigerator. Each tube contains 200 µl of a
competent cell solution. The tubes should be handled with gentleness and tranquility. Label the
tubes with your initials. Label one tube 'pLUX’ and the other tube ‘-’. Place the tubes on ice.
2. Allow the tubes to sit in ice for 3 min. Add ~10 µl of pLUX solution to the pLUX tube. This
volume contains about 50 ng of plasmid DNA. Allow the tubes to incubate on ice for 30 min. DO
NOT shake the tubes or agitate the cells.
3. Transfer the tubes to a 42 °C water bath for EXACTLY 2 min. DO NOT shake the tubes or
agitate the cells.
4. Rapidly and gently return the cells to the ice, and incubate for two minutes.
5. Add 400 µl of room temperature LB broth to each of the two tubes. Incubate at 37°C for 50 min.
This will allow the cells to express the ampicillin resistance before they are exposed to the
ampicillin.
6. Using a new tip, and a freshly sterilized dally rod, use the spread-plate technique to plate 100 µl
of your '-' tube (the one that you DIDN't add plasmid to) on one of your LB + ampicillin plates.
Label the plate on the bottom (lids can fall off).
7. Using a new tip, and a freshly sterilized dally rod, use the spread-plate technique to plate 100 µl
of your pLUX onto an LB plate. Also plate 100 µl of your 'pLUX' tube onto an LB + ampicillin
plate.
Results:
Plate
Tube
LB +Ampicillin
LB
pLUX
pLUX
LB +Ampicillin
'-' (negative control)
LB + ampicillin
transformed cells
LB + ampicillin
untransformed cells
# of Colonies
LB
transformed cells
In the circles above, make a drawing indicating the number and arrangement of your colonies.
Indicate which colonies, if any, are bioluminescent.
Analysis.
1. What happened when you plated 100 µl of untransformed cells onto LB+ampicillin? What does
this indicate?
2. What happened when you plated 100 µl of transformed cells onto LB+ampicillin? How does
this compare with your result when you plated 100 µl of untransformed cells onto LB+ampicillin?
What does this indicate?
3. What happened when you plated 100 µl of transformed cells onto LB? How does this compare
with your result when you plated 100 µl of transformed cells onto LB+ampicillin? What does this
indicate?
4. Do the colonies on the LB plate exhibit bioluminescence? Why or why not? Do the colonies
on the LB+ampicillin exhibit bioluminescence? Why or why not?
5. What is a bacterial colony? How do these colonies form?
6. Cells in nature undergo transformation. What kinds of advantages might this offer to a
population of cells? What are the dangers involved?
7. Many farmers routinely include small amounts of antibiotics in the feed that is given to their
animals. Draw a flowchart describing the steps by which such a practice might lead to an increase
in the infection of humans by antibiotic-resistant strains of bacteria.