Transcript Transformation_Slides_000

Transformation Slides
Cellular organelles targeted with FPs
Human cell stained with two different
fluorescent proteins to visulalize
cytoskeletal components. Transfected
with GFP-tubulin / mCherry actin
(Ben Giepmans)
C Elegans transfected with GFP
tubulin construct (Susan Kline)
Transcription / Translation
Campbell
What is Transformation?
Bacterial
chromosome
Uptake of foreign DNA,
often a circular plasmid
Plasmid
Bacterial
chromosome
Plasmid
Bacteria now express cloned fluorescent protein
(transcription of gene and translation of mRNA to
protein at ribosomes).
Allow bacteria to grow for 1-3
days on plate with ampicillin.
What is a plasmid?
•
A small circular piece of DNA
that replicates separately from
the main bacterial chromosome
•
Originated in bacteria to allow
survival in specific
environmental conditions
•
May express antibiotic
resistance gene or be modified
in the lab to express proteins of
interest
How are plasmids engineered?
DNA Plasmid Vector
Host DNA fragments
(i.e. coral or jellyfish
FP coding DNA)
Ligate (paste) fragments
into cut DNA vector
Cut genomic
DNA into
fragments
+
Cut plasmids
open with
restriction
enzymes
End result: Plasmid
containing FP gene
FP transformation procedure
1. Suspend bacterial colonies in CaCl2
2. Add plasmid DNA
3. Place tubes on ice for 10 min
4. Heat-shock at 42°C for 45 seconds &
place on ice again for 2 min
5. Plate out bacteria
READ THE INSTRUCTIONS AT YOUR LAB
TABLE!
Materials Checklist

1 tube of CaCl2 on ice

2 empty microtubes

1 waterproof pen

4 disposable transfer pipette (or you can use p20’s with yellow tips)

Innoculating loops OR sterile tips

2 cotton swabs

tape for sealing plates after innoculation

1 LB plate

2 LB/AMP plates

Ice bucket (cup with ice and water)

One tube of plasmid labeled either PM1 or PM2 on ice.

Class or lab station waste containers
Why calcium chloride?
Ca++
Ca++
O P O
O
• Transformation solution = CaCl2
• Positive charge of Ca++ ions shields
negative charge of DNA
phosphates so the plasmid DNA
can more easily move through the
cell membrane
O
CH2
Base
O
Sugar
O
Ca++
O P O
Base
O
CH2
O
Sugar
OH
Why ice and heat?
•
Incubate on ice slows fluid
cell membrane
•
Heat-shock increases
permeability of membranes
Why Ampicillin?
• Ampicillin
inhibits cell growth. Only cells that can
inactivate the ampicillin around them will grow.
• Ampicillin resistance
fluorescent protein gene
is tied to (expressed with) the
• Ampicillin is a selection mechanism that only allows
transformed bacteria to grow on the plate
How are Fps used by scientists?
•
Researcher wants to study
a protein of interest,
perhaps a protein that is
part of a pathway such as
respiration.
•
But how does the
researcher visualize the
protein and find out where
in the cell it is expressed?
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP4.htm
How are Fps used by scientists?
•
The FP gene is inserted
into the plasmid right after
the gene for the protein,
before the stop codon.
•
The protein of interest AND
the FP are translated
together.
•
The FP can be seen and
measured, even though the
protein of interest cannot
be seen. Anytime the
protein of interest is made
in the cell, the FP will also
be made.
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP4.htm