AP Bio Lab 8: Transformation We will start on TUESDAY! All the
following information can be found at:
http://phschool.com/science/biology_place/lab bench/lab6/intro.html
Homework: Review all the information on this website, and complete
self-quizzes for understanding of lab procedure and results.
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Key Concepts I: Bacterial Transformation Genetic transformation
occurs when a host organism takes in foreign DNA and expresses the
foreign gene. In this part of the lab, you will introduce a gene
for resistance to the antibiotic ampicillin into a bacterial strain
that is killed by ampicillin. If the susceptible bacteria
incorporate the foreign DNA, they will become ampicillin
resistant.transformation
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Bacterial Colonies The bacterium you use in your laboratory
activity is Escherichia coli, which has been grown in a petri dish
on Luria Broth (LB) agar. Each colony in the petri dish is made up
of millions of individual cells.
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E. coli Bacteria Escherichia coli is the most common bacterium
in the human gut. It has been extensively studied in the laboratory
and is an important research organism for molecular biology. E.
coli reproduce very rapidly; a single microscopic cell can divide
to form a visible colony with millions of cells overnight. Like all
bacteria, E. coli has no nuclear envelope surrounding the bacterial
chromosome and thus no true nucleus. All of the genes required for
basic survival and reproduction are found in the single chromosome.
Some E. coli cells also contain plasmids, small DNA molecules that
carry genes for certain specialized functions, including resistance
to specific drugs.
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Plasmids Plasmids are circular pieces of DNA that exist outside
the main bacterial chromosome and carry their own genes for
specialized functions. In genetic engineering, plasmids are one
means used to introduce foreign genes into a bacterial cell. To
understand how this might work, consider the plasmid below.
Plasmids
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Plasmids cont. Some plasmids have the amp R gene, which confers
resistance to the antibiotic ampicillin. E. coli cells containing
this plasmid, termed "+amp R " cells, can survive and form colonies
on LB agar that has been supplemented with ampicillin. In contrast,
cells lacking the amp R plasmid, termed "amp R " cells, are
sensitive to the antibiotic, which kills them. An
ampicillin-sensitive cell (amp R ) can be transformed to an
ampicillin-resistant (+amp R ) cell by its uptake of a foreign
plasmid containing the amp R gene. To transform cells, you first
need to make them competent to take up extracellular DNA.
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Recombinant DNA
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Competent Cells E. coli cells are more likely to incorporate
foreign DNA if their cell walls are altered so that DNA can pass
through more easily. Such cells are said to be "competent." Cells
are made competent by a process that uses calcium chloride and heat
shock. Cells that are undergoing very rapid growth are made
competent more easily than cells in other stages of growth. The
growth rate of a bacterial culture is not constant. In the early
hours (lag phase), growth is very slow because the starting number
of dividing cells is small. This is followed by a time of rapid
cell division known as the log phase. The actual length of each
phase depends on the temperature at which the cells are incubated.
In this lab, you will start with cells that should be in the log
phase.
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Design of the Experiment I You now have an understanding of how
cells are prepared for transformation. Before beginning the
experiment, it's important to review the basics of sterile
procedure. 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 laboratory, it is important
that you not contaminate your work with any foreign bacteria or
expose yourself to potentially hazardous bacteria. The chart below
summarizes the basics of sterile procedure.
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Go to website http://phschool.com/science/biology_place/la
bbench/lab6/design1.html
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Transformation Procedure In your laboratory, you use plasmids
that carry the amp R gene to transform E. coli cells that lack this
gene. The illustration below outlines the major steps in this
procedure. You also prepare a second group of E. coli cells as a
control to verify that E. coli will not grow on agar with
ampicillin unless it is transformed, and that nothing in the
procedure itself affects the survival of E. coli. The procedure is
the same for both groups of cells except in step 2, where you add
amp R plasmids to the experimental cells but not to the control
cells.
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Closer Look: Transformation Procedure: Step 1
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Closer Look: Transformation Procedure: Step 2
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Closer Look: Transformation Procedure: Step 3
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Closer Look: Transformation Procedure: Step 4
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Closer Look: Transformation Procedure: Step 5 The cells are
incubated for 24 hours
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Closer Look: Transformation Procedure: Step 6
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Analysis of Results I If there is no ampicillin in the agar, E.
coli will cover the plate with so many cells it is called a "lawn"
of cells. Only transformed cells can grow on agar with ampicillin.
Since only some of the cells exposed to the amp R plasmids will
actually take them in, only some cells will be transformed. Thus
you will see only individual colonies on the plate. If none of the
sensitive E. coli cells have been transformed, nothing will grow on
the agar with ampicillin.
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Analysis of Results I
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Homework due tomorrow Review Transformation Process on this
website, and complete self-quizzes for understanding of lab
procedure and results. http://phschool.com/science/biology_place/la
bbench/lab6/trananim.htm
http://phschool.com/science/biology_place/la
bbench/lab6/trananim.htm l
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Tuesday Start Transformation Lab 1.Complete DNA transformation,
set up plates 2.Homework answer Analyzing Results Questions .
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Wednesday gather lab data 1.Gather & Record data 2.Answer
Analyzing Results Questions 1-5 on pg. S106 in lab booklet
3.Calculate Transformation Efficiency, pg.S106-S108 4.Homework
Complete Evaluating Results Questions 1-5 on pg.S109 in lab
booklet