Jonathan Khan March 2, 2011

Ms. Crowley AP Biology Crime Scene, Transformation of Bacteria, and Gel Electrophoresis/DNA Fingerprinting Labs

Crime Scene Lab

Student Questions:

1. What are the three components of DNA?

a. The three components of DNA are:

i. 5-Carbon sugar deoxyribose

ii. Phosphate group

iii. Nitrogenous (nitrogen containing compound) base

2. What is a restriction enzyme?

a. A restriction enzyme or restriction endonuclease is an enzyme whose function is to hydrolyze

DNA into fragments. The enzyme works by recognizing certain base pair sequences, and cuts only
at those sequences.

3. How does a restriction enzyme recognize where to cut DNA?

a. A restriction enzyme recognizes where to cut DNA because it is very specific to where it will cut
the DNA. The enzyme will only recognize a certain nucleotide sequence, and will only cut the
DNA at that sequence.

4. Why would a bacteria contain a restriction enzyme?

a. A bacteria would contain restriction enzymes as a means of protection against invading viruses, so
that they are not successful in taking over the cell. The endonuclease is intended to cut up foreign
DNA as it enters the cell, it acts as a “cellular immune system within the bacteria.

5. Would a restriction enzyme cut up bacterial DNA? Why? Why not?

 a. A restriction enzyme would not cup up bacterial DNA, this is because the bacterial DNA is
methylated at the endonuclease binding sites, which prevents the bacterial DNA from being cut up.

6. What is a buffer?

a. A buffer is a solution containing:

i. A weak acid and a salt of its conjugate base.

ii. A weak base and a salt of its conjugate acid.

b. A buffer functions by effectively maintain the pH of a solution, by neutralizing any extra H+ pr

OH- which may disassociate.

7. What does electrophoresis do?

a. Electrophoresis is the processes used to separate DNA fragments. The fragments form bands as
they move throughout the gel, and a pattern which can be used to determine the length of each
fragment is formed.

8. Why does DNA move through an agarose gel?

a. DNA moves through an agarose gel because it is negatively charged due to the phosphate groups
which make up part of its backbone. The negatively charged fragments of DNA move through the
gel from the negative end to the positive end with the flow of the applied electrical current. The
DNA will move through the pores in the gel.
Lab Language:

• Palindrome:

o A palindrome is a region of DNA in which the sequence of nucleotides is identical with an

inverted sequence in the complimentary strand.

• Incubate/Incubation:

o Incubation is the process of maintaining a set of controlled environmental conditions in order for a
bacterial or tissue culture to develop.
• Solution:

o A solution is a homogeneous mixture of two or more substances.

 The solvent is the substance in which the other dissolves into.

 The solute is the substance being dissolved.

• Buffer:

o A buffer is a solution containing:

 A weak acid and a salt of its conjugate base.

 A weak base and a salt of its conjugate acid.

o A buffer functions by effectively maintain the pH of a solution, by neutralizing any extra H+ pr

OH- which may disassociate.

• Control:

o The run of an experiment in which the variable is not included.

o This allows for a reliable basis for comparison for the run of the experiment that is being used to
test a variable.

• Positive Control:

o A positive control indicates that the basic conditions of the experiment were able to produce a
positive result.

• Negative Control:
 o A negative control indicates that the basic conditions of the experiment were not able to produce a
positive result.

• Digest:

o Digestion if the process of softening or disintegrating be chemical action, heat or moisture.

• Aliquot:

o A portion of a whole especially one of two samples of something that have the same volume or
mass.
 Transformation Lab

Lesson 2 Review Questions:

1. On which of the plates would you expect to find bacteria most like the original non-transformed E.coli
colonies you initially observed? Explain your predictions.

a. Bacteria most like the original non-transformed E.coli colonies initially observed would most
likely be found on the LB –pGLO plate. This is because this plate did not have the plasmid which
means that any growth of bacteria would be normal bacterial growth similar to the initial non
transformed control. These colonies would be found in this plate, because it did not have any
antibiotic on it which would kill the non transformed bacteria.

2. If there are any genetically transformed bacterial cells on which plate(s) would they most likely be
located? Explain your predictions.

a. Any genetically transformed bacterial cells would be located in the LB/Amp +pGLO, and on the
LB/Amp/Ara +pGLO plates. The ampicillin containing medium on this plate would kill all bacteria
 except any that were transformed and became antibiotic resistant enabling them to grow on such a
hostile medium.

3. Which of the plates should be compared to determine if any genetic transformation has occurred? Why?

a. The LB/Amp +pGLO plate and the LB/Amp –pGLO plates should be compared to determine if
any genetic transformation has occurred. Growth on the LB/Amp +pGLO plate would indicate the
success of the uptake of the plasmid. No growth on the LB/Amp -pGLO plate would indivate that
the original bacteria were not already resistant to ampicillin. By this demonstration, the conclusion
could be made that the growth on the LB/Amp +pGLO plate is due solely to the successful uptake
of the plasmid by the bacterial cells.

4. What is meant by a control plate? What purpose does a control serve?

a. A control is kept constant to allow for some form of comparison with the experimental (variable)
group. In this lab, the control group (LB/Amp -pGLO plate) allowed for the observation of the
successful uptake of the plasmid by the bacterial cells. By there being no growth on the LB/Amp –
pGLO plate, it indicates that the bacteria were not already ampicillin resistant, and that the only
way in which they became antibiotic resistant, was through the successful uptake of the plasmid by
the bacteria.

Lesson 3 Data Collection and Analysis:

1. Which of the traits that you originally observed for E.coli did not seem to become altered?

a. Of the traits that were initially observed in E.coli ones that did not seem to become altered are:

i. Reproduction (growth):

1. LB –pGLO, LB +pGLO plate, LB/Amp +pGLO plate, and the LB/Amp/Ara

+pGLO plates all had growth.

ii. Metabolic Activities:

 1. All except for the LB/Amp –pGLO plate reproduced successfully, and there was an
abundance of colonies.

iii. Respiration:

1. All except for the LB/Amp –pGLO plate reproduced successfully, and there was an
abundance of colonies.

2. Of the E.coli you originally noted, which seem to now be significantly different after performing the
transformation procedure?

a. Of the traits that were initially observed in E.coli ones that seemed to become signifigantly altered
after the transformation procedure are:

i. Resistance to ampicillin:

1. Bacteria on the, LB +pGLO plate grew in the presence of ampicillin.

2. All bacteria on the , LB –pGLO plate died showing that they were not initially
resistant to ampicillin.

3. If the genetically transformed cells have acquired the ability to live in the presence of antibiotic ampicillin,
then what might be inferred about the other genes on the plasmid you used in your transformation
procedure?

a. If the bacteria have acquired the ability to live in the prescence ofantibiotic ampicillin, (ampicillin
resistance), the the other genes located on the plasmid were also uptake by the bacterial cell. This
si shown by the glowing bacteria on the , LB/Amp/Ara +pGLO plate. Of which bacteria were
exposed to the enzyme arabinose, and then exposed to ultraviolet light, which made the bacteria
glow.
4. From the results that you obtained, how could you prove that the changes that occurred were due to the
procedure that you performed?

a. The control plate, LB/Amp –pGLO plate, on which the untransformed bacteria did not grow (due
to the presence of ampicillin [the cells were not ampicillin resistant]) proves that the bacteria were
not originally resistant to the ampicillin. Growth on the, LB/Amp +pGLO plate was therefore due
to the successful transformation if the plasmid.
Lesson 3 Data Collection and Analysis:

If a fluorescent green color is observed in the E.coli colonies then a new question might well be raised,
“what are the tow possible sources of fluorescence within the colonies when exposed to the UV light?”

1) Bacteria already had the gene for fluorescence, which allowed it to glow in the prescence of ultraviolet
light.

2) The gene for fluorescence was located on the plasmid uptaken by the bacteria.

1) Recall what you observed when you shined the UV light into a sample of original pGLO plasmid DNA
and describe your observations.

a. Although we did not do this in class, the plasmid DNA should not glow, because it is not the
DNA that is glowing in the bacterial cells, it is the product of the DNA [the protein] which
causes the glowing in the presence of ultraviolet light.

2) Which of the two possible sources of fluorescence can now be eliminated?

a. The assumption that the bacteria already had a gene [before transformation] which allowed it to
fluorescence can now be eliminated, because when placed under ultraviolet light, the bacterial
cells on the LB –pGLO plate did not glow.
 3) What does this observation indicate about the source of fluorescence?

a. This observation indicates that the source of the fluorescence came from the protein produced
by the DNA contained in the plasmid that was uptaken by the bacterial cells.

4) Describe the evidence that indicates whether your attempt at performing a genetic transformation were
successful or not successful.

a. The LB/Amp –pGLO plate, which had no growth showed that the original bacteria were not
resistant to ampicillin,. The LB/Amp + pGLO plate which had bacterial growth in the presence
of ampicillin, proved that the transformation was successful, since the bacteria grew in the
presence of ampicillin, which should kill all of the nonresistant bacterial cells.

Lesson 3 Review Questions:

Look again at your four plates. Do you observe some E. coli growing on the LB plate that does not contain
ampicillin or arabinose?

A lawn of growth is observed on the LB plate that does not contain ampicillin or arabinose.

1) From your results, can you tell if these bacteria are ampicillin resistant by looking at them on the LB
plate? Explain your answer.

a. From my results, it is impossible to tell if these bacteria are ampicillin resistant by just
looking at them on the LB plate.

2) How would you change the bacteria’s environment—the plate they are growing on—to tell if they are
ampicillin resistant?
 a. To tell if the bacteria are ampicillin resistant, change the environment to one of the
mediums containing ampicillin. If the bacterial are ampicillin resistant, they wil grow on this
medium. If not, then there will be no growth on this plate.

3) Very often an organism’s traits are caused by a combination of its genes and its environment. Think
about the greed color you saw in the genetically transformed bacteria:

a. What two factors must be present in the bacteria’s environment for you to see this green
color?

i. The two factors that must be present for the bacteria to demonstrate this green color, are:

1. Ultraviolet light shone on the bacteria.

2. The presence of arabinose sugar.

b. What do you think each of the two environmental factors you listed above are doing to
cause the genetically transformed bacteria to turn green?

i. What each of the two environmental factors listed above is doing to cause the genetically
transformed bacteria to turn green is:

1. The arabinose sugar is allowing the bacteria to glow in the presence of ultraviolet
light that activates the glowing.

c. What advantages would there be for an organism able to turn off or on particular genes in
response to certain conditions?

1. The advantages there be for an organism able to turn off or on particular genes in
response to certain conditions are that the bacteria could adapt to changing
environments and climates enabling it to survive in hostile environments.
Student Questions:

• What is a plasmid?

o A plasmid is a monoploid circular molecule of extrachromosomal DNA.

• Is a plasmid large or small when compared to chromosomal DNA?

o A plasmid is very small when compared to chromosomal DNA.

• Where did the first plasmids come from?

o The first plasmids came from prokaryotic bacteria.

• How are plasmids important to bacteria in nature?

o Plasmids are important to bacteria in nature in that they can have advantageous pieces of DNA
which the bacteria can utilize in stressful environments.

• How do researchers use plasmids?

o Researchers use plasmids in genetic engineering to insert pieces of foreign DNA into a cell so that
the effects of a certain gene not found in the organism, but contained within the plasmid can be
effectively studied in the organism.

• How is a recombinant plasmid made?

o A recombinant plasmid is made by:

• Isolate:

• Plasmid DNA

• DNA containing gene of interest

• DNA of plasmid and DNA of interest gene are cut using the same endonuclease. Thus
producing identical sticky ends.
 • DNA of gene of interest and DNA of plasmid are combined producing a recombinant
plasmid.

• Why do bacterial cells need to be competent?

o Bacterial cells need to be competent because it is only during the time when they are competent,
that they can uptake foreign DNA such as plasmids through the cell membrane. Essentially
becoming competent usually involves the weakening of the cellular membrane (phospholipid
bilayer).

• Describe the process which makes cells competent?

o Cells can be made competent by the addition of Ca2+ salts, and using heat shocks.

• What is transformation?

o Transformation is the uptake of foreign DNA by a cell. In a transformation, genes are usually
uptaken through a piece of DNA called a plasmid which is only naturally found in prokaryotic
cells.
Lab Language:

• Incubate/Incubation:
 o Incubation is the process of maintaining a set of controlled environmental conditions in order for a
bacterial or tissue culture to develop.

• Efficiency:

o Transformation efficiency is an indication of how effectively the plasmid entered into the bacteria.

• Procedure/Protocol:

o Is the sequence of steps followed within a scientific experiment.

• Solution:

o A solution is a homogeneous mixture of two or more substances.

• The solvent is the substance in which the other dissolves into.

• The solute is the substance being dissolved.

• Vortex:

o Is the spiral motion of fluid within a confined area that moves materials in the solution/suspension
to the center of the container.

• Control:

o The run of an experiment in which the variable is not included.

o This allows for a reliable basis for comparison for the run of the experiment that is being used to
test a variable.
• Positive Control:

o A positive control indicates that the basic conditions of the experiment were able to produce a
positive result.

• Negative Control:

o A negative control indicates that the basic conditions of the experiment were not able to produce a
positive result

• Transformation:

o Is the uptake of foreign DNA by a cell. In a transformation, genes are usually uptaken through a
piece of DNA called a plasmid which is only naturally found in prokaryotic cells.

• Plasmid:

o A plasmid is a monoploid circular molecule of extrachromosomal DNA.

• Competent:

o the weakening of the cellular membrane (phospholipid bilayer) to allow for the uptake of foreign
pieces of DNA.

• Aliquot:

o A portion of a whole especially one of two samples of something that have the same volume or
mass.