Transcript Transformation Lab

TRANSFORMATION
Intro to Lab #8
Figure 20.2
Bacterium
1 Gene inserted into
plasmid
Bacterial
Plasmid
chromosome
Recombinant
DNA (plasmid)
Cell containing gene
of interest
Gene of
interest
2 Plasmid put into
bacterial cell
DNA of
chromosome
(“foreign” DNA)
Recombinant
bacterium
3 Host cell grown in culture to
form a clone of cells containing
the “cloned” gene of interest
Protein expressed from
gene of interest
Gene of
interest
Protein harvested
Copies of gene
Basic
research
on gene
4 Basic research
and various
applications
Basic
research
on protein
Gene for pest
Gene used to alter
Protein dissolves
Human growth
resistance inserted bacteria for cleaning blood clots in heart hormone treats
into plants
up toxic waste
attack therapy
stunted growth
Transformation
1.
2.
3.
4.
5.
6.
7.
8.
Identify a restriction enzyme that will cut out
gene of interest and cut open the plasmid
Isolate DNA from 2 sources (making sure the at
the plasmid has a genetic marker)
Cut both types of DNA with the same restriction
enzyme
Mix the 2 types of DNA to join them through
complementary base pairing
Add DNA ligase to bond DNA covalently
producing Recombinant DNA
Incubate bacteria at 42 C with calcium chloride;
bacteria become competent / permeable - so
that the bacteria will take in the plasmid
(TRANSFORMATION)
Use a genetic marker to identify bacteria with the
recombinant plasmid
Clone bacteria
Pre-Lab Inquiry

You work on a team in a research lab. You have
three separate plasmids in your lab, but the labels
have come off of the tubes and you no longer know
which plasmid is which. For the time being, you will
refer to the plasmids are plasmids 1, 2, and 3. You
know the following:
 One
plasmid has kanamycin-resistant gene.
 Two plasmids have an ampicillin-resistance gene.
 In addition to having an antibiotic-resistance marker,
one plasmid also codes for the gene for green
fluorescent protein (GFP), a protein from the
bioluminescent jellyfish Aequorea victoria.
Pre-lab Inquiry


Bacteria that produce the green fluorescent protein look
green under white light and fluoresce under ultraviolet
light
Assume that each research team has access to the
following materials and is responsible for identifying
one of the three plasmid. (Note: you will not actually
use any materials during this pre-lab inquiry). Before
planning your experiment, answer the following
questions to ensure that you understand some of the
basic concepts involved in the lab. Some of these
questions are specifically designed to help you think
about setting up your experiment.
Materials Available



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Starter plates (containing E. coli in rapid growth)
Kanamycin plates
Ampicillin plates
LB plates (plates contain Luria Broth/no antibiotic)
Plasmids 1, 2, and 3
Transformation agents, tubes, and equipment
(inoculating loops, calcium chloride, ice, 42C water
bath, incubator)
Questions

1. What is a plasmid?
 Small,
usually circular, extra-chromosomal piece of
DNA that exits in nature in some bacteria and yeasts.
They can be transferred between organisms. In the lab
they can be used to manipulate and introduce DNA of
interest into bacterium.

2. What is transformation?
 The
uptake of exongenous, naked DNA by a cell. The
newly adopted DNA can become a heritable part of
the cell’s genetic material.
Questions

3. Why is naturally occurring transformation
beneficial to bacteria?
 It
provides them with access to new genetic material,
which increases their chances of being able to adapt to
the environment.

4. Why is transformation useful to research
scientists?
 It
enables them to introduce foreign DNA into bacteria.
This allows them to further study and work with genes
on the plasmid DNA and the proteins that the genes
code for.
Questions

5. Should you plate some of your transformed bacteria
onto plates with antibiotic? Why or Why not?


Yes – you will select for/isolate bacteria that have been
transformed with the plasmid with the resistance gene
6. 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?
Explain.

You would see a lawn of bacteria (the plate would be
covered). This tells you that the bacteria survived the
transformation process. When compared to the antibiotic
plates it gives an indication of how many bacteria were
transformed
Questions

7. 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 combination is taken through
a few more steps to make the bacteria take up DNA.
In your experiment, should you treat a tube of bacteria
that you do not add plasmid to exactly as you do the
tube of bacteria that you will transform? Why or why
not?

This provides a control demonstrating that the antibioticresistance colonies that appear on the plate are a result of
the plasmid being taken up by the cells. If no plasmid, then
there should be no growth on antibiotic plates.
What would you expect?
LB AMP plates
plasmid
Amp
Kan
Amp & GFP
+ plmd
- plmd
LB Kan plates
+ plmd
- plmd
LB plates
+ plmd
- plmd
Color when
present
What would you expect?
LB AMP plates
LB Kan plates
LB plates
Color when
present
plasmid
+ plmd
- plmd
+ plmd
- plmd
+ plmd
- plmd
Amp
25 -100
colonies
0
colonies
0
colonies
0
colonies
Lawn
Lawn
yellow
Kan
0
colonies
0
colonies
25-100
colonies
0
colonies
Lawn
Lawn
Yellow
Amp & GFP
25-100
colonies
0
colonies
0
colonies
0
colonies
Lawn
Lawn
Yellow/gre
en or green
What you need…
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2 LB plates
2 LB/Kan plates
2 LB/Amp plates
2 sterile transformation tubes & rack
Container with crushed ice
3 sterile inoculating loops
6 sterile transfer pipets
Waste container
3 mL vial of LB
3 mL vial of CaCl2 (on ice)
Basic Outline of the Lab
1.
Label 1 tube + / 1 tube –
2.
Add CaCl2, place on ice
3.
4.
5.
6.
7.
8.
Use sterile loop to transfer E. coli
to both tubes (no agar!) return to
ice
Use sterile loop to add loopful of
plasmid (1, 2, or 3) to + tube
Ice for 15 min & label 1of each
plate “+” and 1 of each “-”
Heat shock (42C for 90 sec)
Add luria broth & sit at room temp
5 to 15 minutes
Spread bacteria on plates