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Bacterial Transformation
Pre/PostTest
1. Define the following terms:
a. gene- segment of DNA which codes for one protein
b. plasmid-circular bacterial chromosome
c. protein- polymer made of amino acids
d. gene expression- the translation of a gene into a protein product
e. genotype- the genetic makeup of an organism
f. phenotype- the outward expression of genes of an organism
2. To genetically transform an entire organism, you must insert the new gene into
every cell in the organism. Which organism is better suited for genetic
transformation- one composed of many cells or one composed of a single cell?
Answer: A single celled organism is optimal for total genetic transformation since
it contains only the one cell to take up the one gene.
3. Scientists often want to know if the genetically transformed organism can pass its
new traits on to its offspring and future generations. To get this information,
which would be a better candidate for your investigation, an organism in which
each new generation develops and reproduces quickly, or one which does this
more slowly? Explain your reasoning.
Answer: An organism which reproduces quickly. Fast reproductive rate allows
the scientist to quickly assess whether the new trait has been inherited.
4. What is meant by a control plate? What purpose does a control plate serve?
Answer: A contol plate is a point of comparison (guide) to help you interpret the
experimental results. In this experiment, the control plates were both the –pGlo
plates, for they did not have the plasmid containing the genes for green
fluorescence and ampicillin resistance. The –LB/amp control plate can be
compared to any of the LB/amp plates to show that the plasmid uptake is
necessary for the growth on ampicillin Without the – LB/amp control plate, one
would not know if the colonies on the +LB/amp plate were actually
transformants.
5. Which organism would be the best candidate for total genetic transformation: a
bacterium, a human, a mouse, or an earthworm? Explain your reasoning.
Answer: A bacterium would be the best candidate for total genetic transformation
since it is a single-celled organism which reproduces quickly- every 20 minutes.
6. What is bacterial transformation?
Answer: Bacterial transformation is the uptake of a foreign gene into a bacterial
cell
Pre-Lab Notes for Bacterial Transformation
1. Vocabulary
a. Plasmid- circular chromosomes of bacteria
b. bacterial transformation- the uptake of a foreign gene into a bacterial cell
c. colony- group of bacterial clones growing on the surface of a solid
medium
d. gene- segment of DNA which codes for a protein
e. Protein- polymer of amino acids, differ in sequence, number, and type of
amino acids
f. gene expression- the production of a gene product from process of
translation
g. genome- all the genes present in an organism, encoded in DNA or RNA
h. Genotype-genetic makeup of an organism
i. phenotype – the physical or biochemical charcteristics of an organism,
determined by genes and the environment
j. Antibiotic-anything that kills or inactivates bacteria
k. ampicillin resistance- the ability to survive and/or reproduce in the
presence of the antibiotic, ampicillin
l. Agar- culture medium, made of seaweed extract, for bacteria and other
microorganisms
m. Incubation- the provision of proper conditions for growth and
reproduction
n. control plate- standard of comparison; plate that does not have the
variable you are testing for
o. Fluorescence- emission of light by a substance that has absorbed light or
other electromagnetic radiation of a different wavelength
p. streak plate- the process of inoculating an agar plate with bacteria, using a
sterile loop or glass rod
q. Inoculate- to introduce (bacteria)
r. GFP- green fluorescent protein
s. pGlo plasmid- circular bacterial chromosome containing gene for green
fluorescent protein and enzyme beta lactamase
t. beta lactamase- protein which inactivates ampicillin present in the agar
(the gene for this protein is part of the pGlo plasmid)
u. gene regulation- the ability of genes to be turned on and off, in response to
environmental conditions
v. arabinose- sugar which initiates transcription of the pGlo gene by
promoting the binding of RNA polymerase; when arabinose is not present,
the pGlo gene is not turned on.
Below is a diagram showing the genes involved in pGlo bacterial transformation
By looking at this drawing, what could prevent the bacterial cell from being transformed?
Possible answers: cell wall is not opened, genes are not transferred, not enough plasmid is
in the vicinity of the cell for uptake, plasmid genes are not expressed.
Lesson 2: Review Questions
1. On which of the plates would you expect to find bacteria most like the
original untransformed E.coli colonies you initially observed? Explain
your predictions.
Answer: Bacteria on the –LB will resemble the original untransformed
bacteria, since they were removed from the LB starter plate, plated on
another –LB plate and have no plasmid inserted.
2. If there are any genetically transformed bacterial cells, on which plate(s)
would they most likely be located? Explain your answer.
Answer: The transformed cells would most likely be on either of the +LB
plates, since they contain the inserted pGlo gene. Both of these plates
contain bacteria with the gene for ampicillin resistance, allowing them to
survive on ampicillin.
3. Which plates should be compared to determine if any genetic transformation
has occurred? Why?
Answer: The –LB/amp and +LB/amp should be compared, since the only
difference between the two plates is whether or not they contain the pGlo
plasmid. Cells without the pGlo plasmid do not have the ampicillin
resistance gene, and therefore, won’t be able to survive on ampicillin. The
+LB/amp plate with contained transformed colonies, while the the –
LB/amp plate will not.
4. What is meant by a control plate? What purpose does a control plate serve?
Answer: A contol plate is a point of comparison (guide) to help you
interpret the experimental results. In this experiment, the control plates
were both the –pGlo plates, for they did not have the plasmid containing
the genes for green fluorescence and ampicillin resistance. The –LB/amp
control plate can be compared to any of the LB/amp plates to show that
the plasmid uptake is necessary for the growth on ampicillin Without
the -LB/amp control plate, one would not know if the colonies on the
+LB/amp plate were actually transformants.
Teacher’s Guide to: Lesson 3: Data Collection and Analysis
2. How much growth do you see, relatively, on the plate?
Answer: There should be multiple colonies (around 75) on both the +LB/amp and
+LB/amp/ara, since they received the pGlo plasmid. There should be no growth
on the –LB/amp plate. There should be a lawn of bacteria on the –LB plate, but
no evidence of transformation (glow).
3. What color are the bacteria?
Answer: The bacteria on the +LB/amp and the –LB should be whitish. The
bacteria on the +pglo/LB/amp/ara should appear whitish under normal lighting,
but fluoresce under UV light.
4. Count the number of bacterial colonies that are on each plate (the spots you see).
Answer: There should be approximately 75 bacterial colonies on the two +pGlo
plates. The –LB plate should contain an even lawn of bacteria, too many to count
individual colonies.
Plates
-LB/amp
Observations (Number/color
# of colonies: an even lawn of bacteria, to
many to count individual colonies
Color- whitish
-LB/amp
No bacterial growth
+LB/amp
# of colonies: many individual transformed
colonies
Color- white
+LB/amp/ara
# of colonies- many, approx. 75 colonies
Color- appear white under normal lighting,
fluoresce green when exposed to UV light.
Experimental Group
This depends on the conditions applied.