Download About Transformation In 1928, Frederick Griffith was working on this

About Transformation
In 1928, Frederick Griffith was working on this problem of
finding a vaccine against pneumonia caused by the bacteria
Streptococcus pneumoniae. Here’s what he found:
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In Experiment A, Griffith injected mice with a deadly strain of
pneumococcus that had a capsule. All the mice died.
When Griffith removed the capsule in Experiment B the bacteria
became harmless, and all the injected mice lived.
Griffith then heat-inactivated the deadly capsulated pneumococcus for
Experiment C, and all the injected mice survived.
Finally, in Experiment D, Griffith mixed live harmless pneumococcus
with heat-killed capsulated pneumococcus. While neither of these
strains killed mice alone, they did in combination! All the mice died.
Transform Lab Details
Transformation in Nature: Recipient bacteria and loose
plasmid fragments secrete chemical “competence factors”
that allow the bacteria to take in the plasmid.
Transformation in the Lab: CaCl2 and heat/cold shock
procedures are used to imitate competence factor and rener
the E. coli “competent” or capable of taking in the plasmid
DNA. These steps must be done precisely to achieve good
results.
Our plasmid: PGLO is a
genetically engineered plasmid
that contains three gene groups
of interest:
1. Bacterial genes for
resistance to the antibiotic
ampicillin (Amp).
2. Jellyfish gene for a green
fluorescent protein (GFP).
3. The inducible arabinose
operon, a group of bacterial genes that makes enzymes to
digest the sugar arabinose. The promoter and operator
remain (Ara C), but the other genes have been replaced
with GFP.
An arabinose food source serves as the inducer for the
arabinose operon, and it is needed in order for transformed
bacteria to express the GFP gene.
Ampicillin is used to separate transformed from nontransformed bacteria.
Day1: Do the transformation
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MAKE SURE you understand the steps and procedure you will have just enough time if you are efficient.
Minimum time needed is 35 minutes.
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Each team of 3 students will prepare a control culture and
a transformation culture. Follow the procedure precisely.
At the end of the day, put control and transformed
bacteria tubes onto 4 separate agar plates as follows:
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Ampicillin kills all non-transformed bacteria. Arabinose
induces expression of the GFP gene.
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The transformed bacteria are plated onto ampicillin agar
and ampicillin + arabinose agar. There should be
growth on both plates.
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Control bacteria are plated onto plain agar and
ampicillin agar. There should be growth on the plain
agar but none on the ampicillin agar.
Day 2: Analyze your results
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When you put bacteria onto an agar plate, each bacteria
will divide repeatedly, producing a colony of about 1
million identical cloned bacteria. You can see the colony
which appears as a dot on the plate.
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By counting the colonies, you can determine how many
bacteria you put onto the plate. You can also do a little
algebra to determine the number of bacteria per mL.
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You then calculate the number of transformed bacteria
per ìg of plasmid to find the transformation efficiency.
This procedure is explained on your lab handout.
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We will compare the transformation efficiency of two
different strains of bacteria.
TRANSFORMATION PROCEDURE FOR P-GLO
About micropipettes
You MUST USE A TIP when
drawing in liquid, or you will ruin
the VERY EXPENSIVE
micropipette. Use a fresh tip for each
sample.
Used tips are discarded in the special
waste bins (for biological
waste),NOT place in the regular
garbage can!
Micropipettes work on the principle
of air pressure. The plunger can be
calibrated to various volumes. You
will be using a single-volume pipet
that is calibrated to 10 ìL.
There are two stopping points when depressing the plunger.
• The first “stop” is the calibration point. It lets you draw in
10 ìL. To draw in a sample, push to the first stop, insert the
tip into the liquid, then SLOWLY release the plunger. 10 ìL
will be inside the pipette tip.
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The second “stop” is used to expel the sample. To do this,
place the pipette tip against the side of the tube, press down all
the way, and the entire sample will be expelled. KEEP YOUR
FINGER on the plunger until you withdraw your pipette tip
from the liquid drop.