Bacterial Transformation

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Introduction
Bacterial Transformation   Bacterial
transformation is the process by which bacterial
cells take up naked DNA molecules. If the foreign
DNA has an origin of replication recognized by
the host cells DNA polymerases, the bacteria
will replicate the foreign DNA along with their
own DNA.
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E.coli (eshericia coli) is a bacteria that can be
induced to take up foreign DNA. When the DNA is
incorporated into the cell, the genes expressed
by the original bacteria will now be different.
If DNA is the blueprint of the cell, and we have
now changed the blueprint by transformation, then
the cell itself must look different.   In this
lab we take normal bacteria that look white and
we add new DNA that codes for a blue protein. We
know from protein synthesis that if the bacteria
has the DNA that codes for a blue protein the
Ribosomes in the cell will make the blue protein
and the cells will appear blue.
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Procedure
Day One Using Sterile Technique we placed E.coli
bacteria on new petri dishes. The Petri Dishes
have food for the bacteria
Petri Dish
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Sterile Technique
Sterile Technique involves using an open flame to
sterilize the inoculating loop.
An inoculating loop was used to transfer the old
bacteria onto new food (Nutrient Agar)so we could
use fresh bacteria in our experiment.
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We placed our E. Coli in an incubator so they
could grow overnight.
Fresh E. Coli looks white after it has grown
overnight
Cells growing in an incubator
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Day Two
On Day two we set up the Transformation. We
mixed our E. Coli Bacteria with a new DNA
molecule. This DNA molecule codes for a protein
that is blue in color.
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Procedure
Mr. Williams placed some of our Fresh Bacteria in
an Eppendorf Tube like this one below
He also placed the New DNA in a separate Eppendorf
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We Mixed the tubes together to combine the
Bacteria with the new DNA.
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We placed the tubes on ice for 10 Minutes.
This cooled the Bacteria to about 4 0 Celsius
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Then we placed the bacteria in a Hot Water bath
(42 0C) for 90 seconds.
This Heat Shock helps to open the cell
membranes of the bacteria so they will accept the
new DNA and be transformed.
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We added a Recovery Broth to the cells to help
them Heal from the heat shock.
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We incubated the cells at 37 0 C for ½ hour.
E. Coli Normally lives in usso it likes the
warm temperature. Incubating the E. Coli for ½
hour gave the DNA time to get into the cells.
Warm Water Bath where Bacteria were kept for 1/2
hour
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The bacteria were then streaked onto new Petri
Dishes using Sterile Technique.
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Once again the Bacteria were left in an incubator
overnight to grow.
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Day Three
When we looked at the bacteria on Day Three they
looked like the picture below
What happened ?
Now complete the questions in the lab packet and
do your lab write up.