Transcript Key Concepts I: Bacterial Transformation

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.
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.
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.
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.
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 cont.
• Some plasmids have the ampR gene, which confers
resistance to the antibiotic ampicillin. E. coli cells
containing this plasmid, termed "+ampR" cells, can survive
and form colonies on LB agar that has been supplemented
with ampicillin. In contrast, cells lacking the ampR plasmid,
termed "–ampR" cells, are sensitive to the antibiotic, which
kills them. An ampicillin-sensitive cell (–ampR) can be
transformed to an ampicillin-resistant (+ampR) cell by its
uptake of a foreign plasmid containing the ampR gene.
• To transform cells, you first need to make them competent
to take up extracellular DNA.
Recombinant DNA
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.
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.
Go to website
• http://phschool.com/science/biology_place/la
bbench/lab6/design1.html
Transformation Procedure
• In your laboratory, you use plasmids that carry the
ampR 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 ampR plasmids
to the experimental cells but not to the control cells.
Closer Look: Transformation
Procedure: Step 1
Closer Look: Transformation
Procedure: Step 2
Closer Look: Transformation
Procedure: Step 3
Closer Look: Transformation
Procedure: Step 4
Closer Look: Transformation
Procedure: Step 5
• The cells are incubated for 24 hours
Closer Look: Transformation
Procedure: Step 6
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 ampR 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.
Analysis of Results I
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
• l
Tuesday  Start Transformation Lab
1. Complete DNA transformation, set
up plates
2. Homework  answer Analyzing
Results Questions ….
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