Download pGLO/amp Bacterial Transformation Lab

Name ________________________________________________ Date ___________ Hour _______________
pGLO/ampR Bacterial Transformation Lab
PURPOSE: To gain an understanding of the techniques of culturing E. coli bacteria
and transforming E. coli bacteria using genetic engineering.
INTRODUCTION:
Escherichia coli: E. coli bacteria are the most common bacteria in the human gut. They helps us digest food
and create Vitamin K. E. coli has been extensively studied in the laboratory and is an important research
organism, mainly because it reproduces very rapidly---a single bacterium can divide and form millions of bacteria
over night!
E. coli has all its required genes found in a single chromosome. Some E. coli cells also
contain plasmids—small DNA molecules that carry genes for specialized functions—
including resistance to specific drugs. Scientists have learned how to put new genes into a
plasmid by cutting open the plasmid with restriction enzymes, inserting a new gene into
plasmid, and then placing it into a bacterium. In this lab, you will be using already
genetically altered plasmids that contain a new gene for resistance to the antibiotic
ampicillin and a second gene called pGLO that will enable us to recognize bacteria that
have picked up the plasmids.
NEW
GENE
PLASMID
Resistance: Resistance is the ability of bacteria and other organisms to not be affected or killed by certain drugs
or chemicals. If the bacterium does not have the ampR gene, it will be called an “ –pGLO/ampR cell”, it will not
be resistant to ampicillin and it will be killed by the antibiotic. If the bacterium has the ampR gene, it will be called
a “ + pGLO/ampR cell”, it will be resistant to ampicillin and will survive to form colonies.
Genetic transformation: Genetic transformation is a process where an organism will be forced to take into its
genome a plasmid containing a foreign gene. In this lab, you will be adding a plasmid with the foreign gene,
ampR, into E. coli bacteria that will transform them into ampicillin resistant bacteria.
CHROMOSOME
Ampicillin-sensitive E. coli cell
PLASMID WITH +pGLO/ampR gene
Ampicillin-resistant E. coli cell
Competency: To transform bacteria cells, the cells need to be made competent or capable of taking up DNA
plasmids. Bacteria will be more likely to take up plasmids if their cell walls are altered to allow the plasmids in
more easily. The bacteria cells will be made competent by a process that uses calcium chloride and heat “shock”.
Bacteria cells are also more competent if they are in a rapid growth stage, so the timing of the transformation will
be critical.
Culturing bacteria: Culturing is the process of growing bacteria in Petri dishes on a gelatin-like substance called
agar. Agar contains nutrients and moisture for bacterial growth and reproduction. In this lab, you will be using
Luria Broth (LB) agar. The bacteria will grow in small “piles” called colonies since they contain millions of
individual bacteria cells. Some of the agar will be laced with the antibiotic ampicillin, to determine if the bacteria
are resistant or killed by the antibiotic. Arabinose (ara) is a simple sugar and is a source of energy for bacteria. If
arabinose is present in the bacterium’s environment, it allows the bacterium to turn on genes, to produce the
enzymes, to digest the arabinose. If arabinose is not present, the bacterium does not turn on these genes, and
thus doesn’t waste energy producing an enzyme that is not needed.
STERILE PROCEDURES
The techniques of sterile procedure apply to any activity in which you work with bacteria or fungi. Since you are
working with E. coli bacteria in this lab, it is important that you not contaminate your work with any foreign bacteria
or expose yourself to potentially hazardous bacteria. The chart on the next page summarizes the basics of sterile
procedure.
ALWAYS
NEVER
Always wash your hands and work surface before
beginning.
Never have food on your work surface.
Always keep the lid of the Petri dish on it or over it
at all times. Microbes are everywhere!
Never lay the lid of the Petri dish or culture tube on the lab
bench.
Always open all sterile tools carefully.
Never touch the end of a tool that touches bacteria.
Always keep hair pulled back and use goggles
when flame is present.
Never throw biohazard materials in the regular trash.
Always wash your hands thoroughly with soap and
hot water before leaving the lab.
Never leave a Bunsen burner
flame unattended.
PRELAB: KNOWLEDGE QUESTIONS
1. Describe E. coli. __________________________________________________________________________
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2. What is a plasmid and what are humans using them for? __________________________________________
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3. What genes have been put in the plasmids that we will be using and what is their function?
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4. What is meant by “antibiotic resistance”? _______________________________________________________
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5. What is the difference between an “ +pGLO/ampR cell” and a “ – pGLO/ampR cell”?
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6. What is genetic transformation? ______________________________________________________________
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7. What is meant by a bacterial cell becoming “competent”? __________________________________________
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8. How will we make the bacterial cells “competent”? _______________________________________________
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9. What is “culturing” bacteria? _________________________________________________________________
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10. What is agar and what kind of agar will we be using in the lab? ____________________________________
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11. What is a bacterial colony and how will it look? _________________________________________________
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12. What is ampicillin? _______________________________________________________________________
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13. What happens in a bacterium if the sugar arabinose is in its environment? __________________________
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14. What happens in a bacterium if arabinose is not present? ________________________________________
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15. If a bacterium does not pick up one of the plasmids, and it is placed on an agar that contains ampicillin, what
should happen to the bacterium? _______________________________________________________________
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16. When do sterile techniques need to be used? __________________________________________________
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PRELAB:
Recall that the goal of genetic transformation is to change an organism’s traits (phenotypes). Before any change
in a trait can be detected, a thorough examination of its natural phenotype must be made. Look at the colonies of
E. coli on your starter plate. List all observable traits that can be described.
Number of colonies:
Size of largest colony (mm):
Size of smallest colony (mm):
Color of colonies:
Distribution of colonies
(locations on plate):
Appearance of colonies
under ultraviolet light:
LAB OVERVIEW:
You are going to be setting up four Petri dishes:
One plate will have E. coli bacteria with no plasmids on ampicillin-free LB agar.
This plate will be labeled:
–pGLO/ampR plasmid + LB agar
The second plate will have E. coli bacteria with no plasmids on LB agar with ampicillin in it.
This plate will be labeled:
–pGLO/ampR plasmid + LB/amp agar
The third plate will have E. coli bacteria with ampR plasmids on ampicillin-free LB agar.
This plate will be labeled: +pGLO/ampR plasmid + LB/amp agar
The fourth plate will have E. coli bacteria with ampR plasmids on LB agar with ampicillin and arabinose.
This plate will be labeled: +pGLO/ampR plasmid + LB/amp/ara agar.
-pGLO/ampR +
LB agar
–pGLO/ampR +
LB/amp agar
+pGLO/ampR +
LB/amp agar
+pGLO/ampR +
LB/amp/ara agar
DATA: Describe and draw each bacterial plate.
PLATE NAME
PRE-DESCRIPTION
- pGLO/ampR
plasmid
+
LB
agar
Plate with E. coli
bacteria with no
plasmids on
ampicillin-free agar.
- pGLO/ampR
plasmid
+
LB/amp
agar
+pGLO/ampR
plasmid
+
LB/amp
agar
R
+pGLO/amp
plasmid
+
LB/amp/ara
agar
Plate with
E. coli bacteria
with no plasmids on
agar with ampicillin.
Plate with E. coli
bacteria with
+pGLO/ampR
plasmids on agar
with ampicillin
Plate with E. coli
bacteria with
+pCLO/ampR
plasmids on agar
with ampicillin plus
arabinose
POST-DESCRIPTION
RESULT
CONCLUSIONS AND INTERPRETATIONS
1. To genetically alter an entire organism, you must insert the new gene(s) into every cell in the organism. Which
organism is better suited for total genetic transformation—earthworm, fish, bacteria, mouse, daisy? Why?
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2. Which plate in your experiment was the control? What is the purpose of a control? _____________________
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3. Of the E. coli traits you originally noted, which now seem to be significantly different after performing the
transformation procedure? _____________________________________________________________________
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4. Explain what happened on the - pGLO/ampR plasmid + LB agar plate and why.
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5. Explain what happened on the - pGLO/ampR plasmid + LB/ampR agar plate and why.
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6. Explain what happened on the +pGLO/ampR plasmid + LB/amp agar plate and why.
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7. Explain what happened on the +pGLO/ampR plasmid + LB/amp/ara agar plate and why.
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8. What two factors need to be present in the bacteria’s environment for them to glow green under the UV light?
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9. What advantage would there be for an organism to be able to turn on or off particular genes in response to
certain environmental conditions? ______________________________________________________________
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