Download Bacterial Transformation Lab

Learning Targets
“I Can . . .”
-Explain what it means for a gene to be expressed.
-Explain the role of plasmids.
-Insert a plasmid into bacteria to observe the
process of transformation as an important
biotechnology process.
-Define bacteria “transformation.”
Bacterial Transformation
Using the pGLO gene
A Study of Gene Expression
Biology and Honors Biology:
Start reading through the
experiment procedures on your
tables so that you are familiar
with what we will be doing today.
Background and Review
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A gene is a piece of DNA that contains the
instructions for making a particular protein; this
protein gives an organism a certain trait
Some genes can be inserted into organisms to
change their traits; this is called . . .
TRANSFORMATION
Anatomy of a Bacteria Cell

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Bacteria contain bits of circular DNA called
plasmids
Bacteria can exchange plasmids freely with one
another and with other sources
Background and Review

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You will be working with a bacteria called E. coli
In order to transform E. coli, you need to grow
LOTS of it
Today we will be placing E. coli on a petri dish
and incubating it at body temperature (37◦C) to
grow colonies.
Each 1mm colony consists of millions of
bacteria cells originating from a single cell that
has divided through mitosis for several
generations
What is a colony?
How do we get colonies?
By Preparing A Streak Plate
Purpose of a Streak Plate
What is aseptic technique?
Be as sterile as possible!
*Wipe the surface you are working on before
and after the lab
*Sterilize your instruments with extreme heat (or
use disposable instruments)
*Open the petri dish lid slightly; do NOT lay the
lid on the table
What is aseptic technique?
Why aseptic technique?
What will you do in lab?

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Green Fluorescent Protein (GFP) causes the
jellyfish species Aequorea victoria to glow in the
dark
This gene will be inserted into bacteria, in the
form of a plasmid, to cause them to glow as well
What will you do in lab?
The pGLO plasmid contains two genes:

1.
2.
The gene for GFP (glowing gene)
The gene to be resistant to ampicillin (an antibiotic)
Two pGLO tubes in lab:
-pGLO= no pGLO plasmid
+pGLO = pGLO plasmid
For the GFP to actually glow, the sugar arabinose must be
present.
LB Petri Dish (-pGLO)

LB is a nutrient that allows E. coli to grow
LB/amp Petri Dish (-pGLO)
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LB is a nutrient that allows E. coli to grow
Amp stands for ampicillin, an antibiotic
What do antibiotics do to bacteria growth?
LB/amp Petri Dish (+pGLO)
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LB is a nutrient that allows E. coli to grow
Contains ampicillin
Contains plasmid with two genes
LB/amp/ara Petri Dish (+pGLO)
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LB is a nutrient that allows E. coli to grow
Contains ampicillin, an antibiotic
Also contains arabinose, a sugar
Contains plasmid with two genes
Review Questions
1.
On which of the plates would you expect to
find bacteria most like the original nontransformed E. coli on your starter plates?
Explain your predictions.
LB –pGLO
LB/amp –pGLO
LB/amp +pGLO
LB/amp/ara +pGLO
Review Questions
2. If there are any genetically transformed
bacterial cells, on which plate(s) would they
most likely be located? Explain your
predictions.
LB –pGLO
LB/amp –pGLO
LB/amp +pGLO
LB/amp/ara +pGLO
Review Questions
3.
Which plates should be compared to
determine if any genetic transformation has
occurred? Why?
LB –pGLO
LB/amp –pGLO
LB/amp +pGLO
LB/amp/ara +pGLO
Review Questions
4. What is meant by a control plate? What
purpose does a control serve?