Download Lab 7 - Bacterial Transformation

Biology 211
Bacterial Transformation with pGLO,
Part 1
OBJECTIVES:
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Practice formulating hypotheses, predictions, and experimental design.
Describe the principles of bacterial transformation.
Explain the procedure for gene transfer using plasmid vectors.
Induce the transfer of the pGLO gene (in a plasmid) into E. coli.
Describe the traits carried by the pGLO gene.
Describe how to activate (“turn on”) the pGLO gene.
Describe how to recognize the transformed cells (from this lab).
Know the terms used in this lab including transformation (in this case transformation does NOT mean
the conversion of a normal cell to a cancerous one), vector, plasmid, fluorescence, antibiotic
resistance, E. coli.
INTRODUCTION:
In this lab you will perform a procedure known as a genetic transformation. Remember that a gene
is a piece of DNA that provides the instructions for making (coding for) a protein that gives an organism a
particular trait. Genetic transformation literally means change caused by genes and it involves the insertion
of a gene(s) into an organism in order to change the organism's trait(s). 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. In bio-remediation, bacteria can be genetically
transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes
are beginning to be treated by gene therapy; that is, by genetically transforming a sick person's cells with
healthy copies of the gene involved in their disease.
You will use a procedure to transform bacteria with a gene that codes for GFP. The
original source of this gene is the bioluminescent jellyfish Aequorea victoria. The gene
codes for a Green Fluorescent Protein that causes the jellyfish to fluoresce and glow in
the dark. Following the transformation procedure, the bacteria express their newly
acquired jellyfish gene and produce the fluorescent protein that causes them to glow a
brilliant green color under ultraviolet light.
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, which creates
the opportunity for them to share these beneficial genes. (Note that the bacteria don’t know that they are
picking up beneficial genes.) This natural mechanism allows bacteria to adapt to new environments. The
recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.
In this lab, we are using a plasmid called pGLO, short for
plasmid – glowing. This unique plasmid we use encodes the
gene for the Green Fluorescent Protein (GFP) and a gene for
resistance to the antibiotic, ampicillin (this gene is called beta
lactamase). The unique plasmid we use encodes the gene for
the Green Fluorescent Protein (GFP) and a gene for resistance to
the antibiotic, ampicillin. The plasmid also incorporates a special
gene regulation system, which can be used to control expression
of the fluorescent protein in transformed cells. The gene for the
Green Fluorescent Protein can be switched on in transformed
cells by adding the sugar, arabinose (ara), to the cells’ nutrient
medium. Selection for cells that have been transformed with the
plasmid DNA is accomplished by growth on antibiotic plates.
Biology 211
Transformed cells will appear white on plates not containing arabinose, and fluorescent green under UV light
when arabinose is included in the nutrient agar.
You will be provided with the tools and a protocol for performing genetic transformation in
Escherichia coli. This transformation procedure involves three main steps.
1) Introduce plasmid DNA to E. coli cells
2) Force the movement of plasmid DNA through the E. coli cell membrane
3) Provide an environment for the cells to express their newly acquired genes
Many species of bacteria have special membrane proteins for the uptake of DNA from the external
environment. E. coli does not appear to have these types of membrane proteins; however, placing E. coli in
a relatively high concentration of calcium ions and performing a procedure called “heat shock” will
stimulate these cells to take up pieces of foreign DNA.
To move the plasmid DNA through the cell membrane you will:
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Use a transformation solution of CaCl2 (calcium chloride)
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Carry out a procedure referred to as heat shock
Note that for transformed cells to grow in the presence of ampicillin you must provide them with nutrients
and a short incubation period to begin expressing their newly acquired genes!
Read the lab exercise and follow the directions carefully. You will do this lab in lab groups of 3-4 students.
Completion of this part of the lab will take 2 lab periods (or 1 lab and 1 class). In the second lab period you
will analyze your results.
PART I : BACTERIAL TRANSFORMATION
Exercise A:
Introduction to Sterile Technique (in lab session)
You will practice using sterile technique, as instructed at the beginning of lab session, before you do
the experiment. When culturing bacteria, you must not introduce other, contaminating bacteria into your
culture. Potentially contaminating bacteria are ubiquitous; they are found everywhere (including on the
bench top and on your hands). It is especially important to keep the inoculation loops, the pipette tips, and
the surfaces of the agar plates must not touch or be touched by any contaminating surface.
Exercise B:
Bacterial Transformation (in lab session)
MATERIALS & PROCEDURES
1. Follow the procedures in the “Transformation Kit-Quick Guide” provided in lab. Be sure to record your
protocol in your lab notebook!!
2. The plates will be incubated for 24-48 hours, and then placed in a refrigerator to slow the growth of the
bacteria. You will than observe the plates in the next lab period to collect your data.
3. Before leaving lab today, make sure that all contaminated items are placed in the biohazard bucket and
take care to properly disinfect your lab bench.
LAB NOTEBOOK CHECK: Your lab notebook entry this week should contain a title and objective for this
lab. As always, you should include detailed procedures. (You may simply tape them in from this
handout if you like.)
Bacterial Transformation with pGLO, Part 2
INTRODUCTION:
Recall that in your last laboratory session, you performed a transformation, hopefully introducing the
pGLO plasmid into E. coli. In this session, we’ll examine our bacterial plates to determine whether or not this
transformation was successful. Fortunately, this is an easy task. The Green Florescent Protein (GFP) emits
green light when excited by UV light. This allows researchers like ourselves to quickly determine whether or
not the protein is being expressed. For this reason, GFP is commonly used in research labs around the world.
Biology 211
PROCEDURE:
1. Retrieve your plates from the incubator or fridge. Count the colonies formed on each plate and record
this result in an organized table in your lab notebook. Note that if you have a very large number of
colonies, you may count either a half or a quarter of the plate and estimate the total plate count from
that number. In some cases, you may have so many growing bacterial colonies on your plate that they
don’t have the space or resources to form distinct colonies; this is referred to as a bacterial “lawn”.
2. Use one of the UV light boxes in the lab to carefully examine your bacterial plates under UV light. To best
see the colonies, you should place the petri dish right side up in the light box, and carefully remove the
lid during viewing. Record your observations in the data table you created for your bacterial counts in
step 1.
3. When you have finished with your plates, please be sure they are placed in a biohazard bucket and that
your work space is properly disinfected.
POSTLAB:
Only one writeup is necessary for both parts
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Date, title, page number
Objective
Materials and methods
Results
o Data table of your results (modeled after the prelab)
Discussion
o Was your genetic transformation successful? How do you know?
o Consider the following two pairs of plates. What do the results obtained from these plates tell
you about your experiment?
1. -DNA LB and -DNA LB/amp
2. +DNA LB/amp and -DNA LB/amp
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After examining your results, would you revise your hypothesis? Why or why not? What is your
revised hypothesis if you have one?
What was the purpose of the experiment?
How could you improve the experiment?
What other things could you test using the techniques you learned in the lab?