Transcript Biotechnology: Bacterial Transformation

Biotechnology: Bacterial
Transformation Lab
How can we manipulate
inheritable information?
You are going to be split into one of
the following groups
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DNA cloning and applications
Different methods used to study DNA
Medical applications of DNA technology
Agricultural applications (plants & animals)
Safety and Ethical issues surrounding the
applications of DNA technology
You will create a poster about your
topic & present it to the class.
Genetic information passed from
parent to offspring via DNA provides
for continuity of life.
• In order for DNA to direct cellular activities it
must first be transcribed.
• Some of the RNA’s are used immediately for
ribosomes or to control other cellular
processes.
• Other RNA’s are translated into proteins that
have important roles in determining
metabolism & development.
• When the DNA of a cell changes, the RNA’s
and proteins they produce often change,
which in turn changes how the cell functions.
• DNA can change in different ways:
– Mutated (spontaneously, environmental effects or
DNA replication error)
– Biotechnologists can cause an intentional change
Biotechnologists have a most powerful
tool:
• They have the ability to transfer the DNA of
one organism into another & make it function
in the new organism.
• With this ability they can make cells produce
novel protein products that the cells did not
make previously.
Fredrick Griffith Experiment
CONCLUSION: The living R bacteria had been
transformed into pathogenic S bacteria by an unknown,
heritable substance from the dead S cells that allowed
the R cells to make capsules.
What they do today…
• Biotechnologists have succeeded in inserting a
gene (Bt) from the bacterium:
– Bacillus thuringiensis
into the corn genome.
• When expressed the Bt gene produces a toxin
that kills caterpillars & controls earworms that
damage corn
Applications for genetic engineering
Human manipulation of DNA has raised several
ethical, social, and medical issues
Working with bacteria
What’s a plasmid?
A plasmid is a small
circular piece of
double-stranded
DNA that has an
origin of replication.
Natural
Transformation
Why is this beneficial
to bacteria?
Gives them more genetic
material which may
increase their chances of
surviving in a changing
environment.
Cells have surface proteins that bind to DNA and bring it into the cell.
Then the orig. DNA and the new DNA are compared. If there is enough
similarities the new DNA will be welcomed. If there isn’t then the new
DNA is digested by the cellular enzymes.
Why is transformation useful to
research scientists?
• Allows for further study or work with genes on
the plasmid DNA and the proteins that the
genes code for.
• Enables researchers to manipulated and study
genes already present in the bacterial
genome.
Should you plate some of your
transformed bacteria onto plates with
antibiotics? Why or why not?
• Yes, this ensures that those ;bacteria that take
up the plasmid will retain it and allows you to
select for those bacteria that have actually
taken up the plasmid.
What would you expect to see if
you plated some of your
transformed bacteria onto a
plate without antibiotic? Would
there be an advantage to doing
this (in terms of understanding
your results)? Explain
• You would see a “lawn”. Yes, this would allow
you to assess cell viability. How viable the cells
are is important to know in the event that
there are no colonies on the plate that does
contain antibiotic.
• In addition, being able to directly contrast the
lawn on the antibiotic-free plate with the
colonies present on the plate with antibiotic is
a good demo. Of how small a % of cells
actually took up the plasmid.
• Contrast also indicates that the antibiotic is
good.
• To transform bacteria with plasmids, technicians
first make the bacteria competent (capable of
taking up DNA) by placing them in calcium
chloride and chilling them.
• Plasmid is then added to the competent bacteria
and the plasmid/bacteria combo. Is taken
through a few more steps to make the bacteria
take up the DNA.
• In your experiment, should you treat a tube of
bacteria that your don’t add plasmid to exactly as
your do the tube of bacteria that you will
transform?
• Why or why not?
• Treating bacteria that you have not added
plasmid to exactly as you do the bacteria that
you add plasmid to, provides a control
demonstrating that the antibiotic-resistant
colonies that appear on the plate are a result
of the plasmid being taken up by the cells.
• In the absence of plasmid, there should be no
antibiotic-resistant colonies present on the
plate.
Let’s work with bacteria!!!!
TOMORROW 
COLONIES
LAWN
Rendering bacteria competent
• Changing the ionic strength of the medium and
heating the cells in the presence of positive ions
(usually calcium).
• This treatment renders the cell membrane
permeable to DNA.
• Recently high voltage has also been used to
render cells permeable to DNA
– electroporation
• Once the DNA has been taken in by the cell, the
use of that DNA by the cell is referred to as
expression
• The expression of the new DNA depends on its
integration with the host DNA.
• Normally scientists want to introduce DNA that is
not similar to the host DNA & therefore the cell
would destroy the introduced DNA.
• Scientists have found a way around this by
introducing the new DNA as plasmid DNA.
– Does not have to be similar to the host DNA
Selecting for transformed bacteria
• 2 problems to overcome:
– Cells containing plasmids reproduce at a slower
rate & the pressure is great to rid themselves of
the plasmid.
• To overcome this there needs to be an advantage
– We have to be able to determine which bacteria
have received the plasmid.
Solutions to the problems
• Scientists use a system involving antibiotics
and genes for resistance of antibiotics.
• In a typical transformation, billions of bacteria
are treated and exposed to plasmid DNA.
• Only a fraction (less than 1 in 1000) will
acquire the plasmid.
• Antibiotic resistance genes provide a means of
finding the bacteria that took in the plasmid.
• The treated bacteria are then exposed to the
antibiotic.
• The bacteria that took in the plasmid will grow
on the antibiotic plate.
• Resistance to an antibiotic is known as a
selectable marker; we can select for cells that
contain it.
• Other marker genes are color marker genes,
which change the color of a bacterial colony.
Green Fluorescent Protein (GFP)
This is an example of a color
marker scientists use.
Color markers used as reporter
molecules
• If you wanted to know what a certain protein
did or where it went you could attach the
color marker.
• Let’s say you wanted to know if a particular
protein had anything to do with making blood
vessels. If the blood vessels started glowing
you would have your answer.
Suppose you have a plasmid that
contains both the gene for GFP and
a gene for resistance to ampicillin
(an antibiotic), how will you be
able to determine if the bacterial
cells have been transformed?
What are you going to do?
• Some bacteria are naturally resistant to
antibiotics, but others are not.
– How could you use LB/agar plates, some E. coli,
and some ampicillin (an antibiotic) to determine
how E. coli cells are affected by ampicillin?
• What would you expect your experimental
results to indicate about the effect of
ampicillin on the E. coli cells?