Transcript of Biotechnology: Bacterial Transformation Lab How can we manipulate inheritable information?
- Slide 1
- Biotechnology: Bacterial Transformation Lab How can we
manipulate inheritable information?
- Slide 2
- You are going to be split into one of the following groups 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.
- Slide 3
- 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 RNAs
are used immediately for ribosomes or to control other cellular
processes. Other RNAs are translated into proteins that have
important roles in determining metabolism & development.
- Slide 4
- When the DNA of a cell changes, the RNAs 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
- Slide 5
- 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.
- Slide 6
- 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.
- Slide 7
- 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
- Slide 8
- Applications for genetic engineering
- Slide 9
- Human manipulation of DNA has raised several ethical, social,
and medical issues
- Slide 10
- Working with bacteria
- Slide 11
- Whats a plasmid? A plasmid is a small circular piece of
double-stranded DNA that has an origin of replication.
- Slide 12
- Natural Transformation 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 isnt then the new DNA is digested by the
cellular enzymes. Why is this beneficial to bacteria? Gives them
more genetic material which may increase their chances of surviving
in a changing environment.
- Slide 13
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- 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.
- Slide 15
- 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.
- Slide 16
- 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
- Slide 17
- 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.
- Slide 18
- 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 dont add
plasmid to exactly as your do the tube of bacteria that you will
transform? Why or why not?
- Slide 19
- 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.
- Slide 20
- Lets work with bacteria!!!! LAWN COLONIES TOMORROW
- Slide 21
- 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
- Slide 22
- 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
- Slide 23
- 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.
- Slide 24
- 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.
- Slide 25
- 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.
- Slide 26
- Green Fluorescent Protein (GFP) This is an example of a color
marker scientists use.
- Slide 27
- 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. Lets 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.
- Slide 28
- 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?
- Slide 29
- 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?