Name: ________________________________Copy___________ Date: ________ Class: _______
Genetic Transformation
The Genetic Creation of Glowing Bacteria
Introduction & Background
During this laboratory exercise you will perform a procedure known as genetic
transformation. Remember that a gene is a piece of DNA which provides the instructions
for making a specific protein through the process of protein synthesis (gene expression).
This protein then gives the organism a particular trait. Genetic transformation literally
means change caused by genes, and involves the insertion of a gene into an organism in
order to change the organism’s trait. Genetic transformation is used in many areas of
biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage
resistance can be genetically transformed into plants. Bacteria have been genetically
transformed with genes enabling them to digest oil spills.
You will be using a procedure to transform bacteria with a gene that codes for a Green
Fluorescent Protein (GFP). The real-life source of this gene is the bioluminescent jellyfish
Aequorea victoria. Green Fluorescent Protein (GFP) causes the jellyfish to glow in the
dark. Following this procedure, the bacteria should express their newly acquired jellyfish
gene and produce the fluorescent protein, which causes them to glow a brilliant green
color under ultraviolet light.
Genetic Transformation: Change caused by genes; inserting the genes from one
organism into another organism
Green Fluorescent Protein (GFP):
The protein created in the jellyfish when the gene for bioluminescence (glowing) is
expressed
In this activity, you will learn about the process of moving genes from one organism to
another with the aid of a plasmid. In addition to one large chromosome, bacteria naturally
contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually
contains genes for one or more traits that may be beneficial to bacterial survival. In
nature, bacteria can transfer plasmids back and forth allowing them to share these
beneficial genes. This natural mechanism allows bacteria to adapt to new environment.
The recent occurrence of bacterial resistance to antibiotics is due to the transmission of
plasmids.
Plasmid:
Circular piece of DNA in a bacterial cell; can be passed from one bacteria to
another easily; usually contains genes for traits beneficial to survival.
LB: Luria Broth; food for the bacteria so that they can multiply quickly (clone
themselves)
During the laboratory exercise you will be inserting two genes into the bacteria. The
gene for GFP which when expressed will cause the bacteria to glow, and the gene for
antibiotic resistance which when expressed will cause the bacteria to NOT be destroyed
by antibiotics. Both of these genes combine and are called pGLO. Even when the gene
for GFP is inserted into the bacteria, it must be “turned on” with a sugar called arabinose.
Antibiotic: drug that kills or prevents the growth of bacteria
Ampicillin: A type of antibiotic that destroys E.Coli under normal conditions
pGLO: GFP gene & Antibiotic Resistance gene
Arabinose: Sugar that “turns on” the GFP gene
You will recognize the flow chart below which represents protein synthesis (gene
expression). Complete flow chart #1 and then use this information to complete flow chart
#2.
1. DNA → RNA → __protein___ → Genetic Characteristic (Trait)
2. DNA → RNA →
Materials
7 sterile loops
6 sterile pipettes
2 small collection tubes
1 floating foam
1 sharpie
Ice bath
Incubator (37^C)
GFP
→ ___bioluminescence (glowing)___
1 petri dish (LB)
2 petri dishes (LB/Amp)
1 petri dish (LB/Amp/Ara)
antibacterial soap
masking tape
Warm water bath (42^C)
Safety
 Wash hands thoroughly before and after lab.
 Wash hands immediately after any handling of the bacteria.
 Report any spills to the teacher immediately.
 Dispose of all contaminated loops, pipettes, and collection tubes in the designated
trash cans.
 For additional information about E.Coli refer to the supplemental poster.
Pre-Lab Questions
1. To genetically transform an entire organism, you must insert the new gene into every
cell in the organism. Would a bacteria or a human be easier to genetically transform?
____________bacteria__________________________________________________
2. List two examples of why bacteria are more easily transformed than a human.
1. Single celled; only need to change the DNA of one organism to change
its traits.
2. Multiply quickly so that we can see a result quickly
Predictions
Four Petri dishes will be used to grow bacteria under different conditions. There will be
abbreviations on each Petri dish to help explain the conditions under which the bacteria
will be grown.
What does each abbreviation mean?
 LB: Luria Broth (bacteria food)
 Amp: Ampicillin (antibiotic that destroys E.Coli)
 Ara: Arabanose (sugar that turns GFP gene on)
 +pGLO: Contains GFP gene and Antibiotic Resistance Gene
 -pGLO: Does not contain “new” genes; no genetic change to bacteria
Use the chart below to
1. Explain the conditions within the Petri dish
2. Hypothesize the outcome of bacterial growth
3. Collect qualitative data following experiment
Petri Dish
Explanation of
Conditions
(Petri Dish
Contents)
*GFP & Antibiotic
Resistance Gene
*Luria Broth
+pGlo/LB/Amp
(bacteria food)
*Ampicillin
(antibiotic)
* GFP & Antibiotic
Resistance Gene
* Luria Broth
+pGlo/LB/Amp/Ara (bacteria food)
* Ampicillin
(antibiotic)
* Arabinose
-pGlo/LB/Amp
-pGLO/LB
* No new genes
* Luria Broth
(bacterial food)
* Ampicillin
(antibiotic)
* No new genes
* Luria Broth
(bacterial food)
Hypothesize
growth on
Petri dishes
Bacteria will grow
but will not glow
because the
arabinose did not
turn on the gene
Bacteria will grow
and glow
Bacteria will not
grow or glow
because the
ampicillin will
destroy the
bacteria without
the resistance
gene being
present
Bacteria will grow
but not glow
because the GFP
gene was not
added
Observation of
Petri dishes
after several
days