Modeling Plasmid Selection
And Authentic Assessment
6-2010
http://biology2020.wikispaces.com/
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With the pGlo Plasmid
Bacteria
are very useful organisms in
genetic engineering.
They

are able to bring in plasmids.
Into which DNA of interest may be
added.
The Problem?
• Finding the bacteria that have taken in
the modified plasmids.
• You can see the bacteria, but not the
plasmid.
• Must look for some visible effect of the
plasmid.
What to look for?
• Growing in the presence of antibiotic.
• The effect of a gene, like the glow of
pGLO
Cut Four Paper Ovals
(or…draw 4 ovals on a large sheet of
paper.)
• Each represents a bacterial cell
Make Four Chromosomes
• Out of four chenille stems
• Bacterial
• Circular
• Give a little twist to prevent confusion with
a nucleus.
• Add to “cells.”
• (Or just draw this chromosome in on paper)
Form your plasmids
Use 1/2 a chenille stem each. Make two plasmids as below.
Twist into ring but leave tails
Regulatory Gene
bla
Ara C
Antibiotic Resistance
Ara B
Ara A
AraD
Structural Genes
GFP
Bla codes for beta lactamase, a
protein which confers antibiotic
resistance.
By the way…great place to teach the operon.
Reserve
Restriction Enzymes
• Plasmids are cut with the same restriction enzyme used to cut the
DNA to be inserted. A restriction enzyme which leaves overhanging
sticky ends is needed for this this procedure. This provides the free
base pairs needed to combine the plasmid DNA with the source DNA.
Restriction
Enzyme Cut from
EcoRI
✤
Procedure:
•
• Open the plasmids you prepared by untwisting
the ½ chenille stem.
• This represents the recognition site for the
restriction enzyme. Remove ara B, araA, and
araD (last 4 beads).
• Add a green bead to represent the GFP (gene
of interest).
Modeling pGlo
LB
-pGLO
LB/AMP
-pGLO
LB/AMP
+pGLO
LB/AMP/ARA
+pGLO
Bacteria Lives?
LB
Yes
-pGLO
LB/AMP
No
-pGLO
LB/AMP
LB/AMP/ARA
Yes Yes
+pGLO
+pGLO
Bacteria Glows?
LB
No
-pGLO
LB/AMP
LB/AMP
LB/AMP/ARA
No
No
Yes
-pGLO
+pGLO
+pGLO
Great Lab for Authentic
Assessment
Let’s try it.
More Authentic Assessment
1. Is this gel in the
chamber correctly?
2. How do you know?
2nd block may
get this!
Provide a ruler and
some semi-log paper.
Ask: How big is
fragment X?
Which is larger , X or
Y?
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Micropipettor
• Have out a micropipettor and a
microtube.
• Instructions:
– Pipet 50 µL of water into the
microtube.
– Close and write your initials on the
top with the permanent marker.
– Place in the box by clicker number.
– Set the micropipettor to the day of
the month you were born on.
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Osmosis
• Provide a scale and weigh boat.
Just use water…but ask…
• This bag has been in this beaker
of distilled water for 30 minutes.
• The initial mass of the bag was
30 g.
• Is the bag hypertonic, hypotonic,
or isotonic to the beaker?
• Which way did water move?
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Fruit Flies
Give sex and eye type.
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Mitosis
Identify
the stage
of mitosis
at the tip
of the
pointer.
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Respiration
Read the
pipette.
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References
• Biotechnology Explorer, pGLO Bacterial
Transformation Kit. BioRad.
http://www3.bio-rad.com/images/pglomap2.gif
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