• Make a new entryEntry 49:pGLO Quiz Review-5/9/12
See attached worksheet
• 1. What are plasmids? What type of
organism generally has plasmids
• Plasmids are circular, double stranded DNA
found in bacteria
Bacterium
• 2.Describe the function of
• a. a restriction enzyme • b. the enzyme ligase -
• Restriction enzymes can cut specific
sequences of DNA— this allows for a
section of DNA to be removed or
inserted into a cell (scissors)
• Ligase (enzyme) can paste pieces of
DNA together– joins the ends where
the restriction enzyme cut (tape/glue
3. Draw a short piece of DNA and show how a
restriction enzyme might cut the DNA
GGATTCC
CCTAAGG
G
CCTAAG
GATTCC
G
4. When you put the paper GFP gene into your
paper plasmid, why did you have to cut the
plasmid at the same sequence of DNA letters
as the uneven ends of the GFP gene ?
This makes sure the ends of the plasmid DNA
and the new DNA will fit together
GGAATT
CCTTAA
GGAATTTGGAATTCCCGAATT
CCTTAAACCTTAAGGGCTTA A
• 5)
Gene
Enhancer
Promoter
Coding sequence
Terminator
THE PLASMID
GFP GENE
AMPICILLIN
RESISTANCE
GENE
(Protects
against AMP)
(gene from jellyfish—will glow
green if have correct
environment/turned on)
• 6. In the pGLO lab, we use a heat shock
(ice – 42˚C – ice) on our bacteria. Why?
• This process opens up the cell to
force in the plasmid.
PLASMIDS WITH GFP GENE AND
ANTIBIOTIC RESISTANCE GENE
PORES
PLASMIDS
PLASMIDS WITH GFP GENE AND
ANTIBIOTIC RESISTANCE GENE
• 7. From the pGLO lab, describe why the –P
Glo, – AMP plate had many colonies of
bacteria, while the –P Glo, +AMP plate has
zero bacteria.
-PGLO -AMP
-PGLO +AMP
The –PGLO –AMP plate had nothing to kill the bacteriaso many bacteria grew
The –PGLO +AMP had AMP—which kills bacteria—and
the bacteria did NOT have resistance since they were
regular bacteria
• 8. From the pGLO lab, describe why the
–
pGLO, +AMP plate had zero bacteria while
the +pGLO, +AMP plate had some colonies of
bacteria.
-pGLO +AMP
+pGLO +AMP
Both plates contain AMP—which kills regular
bacteria (-pGLO)—BUT—the +PGLO bacteria
have resistance to the AMP
9. Which of these plates would have glowing
colonies? Give evidence from our lab. Be sure
to explain WHY since both plates are +pGLO
BOTH PLATES ARE +PGLO, + AMP
THE PLATE ON THE LEFT
WOULD HAVE GLOWING
COLONIES BECAUSE IT
HAS THE DNA/PLASMID
AND THE ARABINOSE
(ENVIRONMENT) WHICH
WOULD SWITCH ON THE
GFP GENE.
THE PLATE ON THE RIGHT
HAS THE DNA-BUT THE
GFP GENE ISN’T
SWITCHED ON—NO
ARABINOSE
10. Based on the results of our lab, how do we
know AMP will kill regular/non-transformed
bacteria?
-PGLO +AMP
The bacteria had no
resistance to the AMP—so
they were all killed
When no AMP was present
the bacteria all survived
-PGLO -AMP
• 11. Based on the results of our lab, how do we
know the plasmid provides antibiotic
resistance?
-PGLO +AMP
+PGLO +AMP
The +PGLO +AMP plate gave evidence because
the bacteria that were given the plasmid survived—
those that did NOT have the plasmid were all killed
12. Based on the results of our lab, how
do we know that just arabinose will not
produce glowing colonies?
+ PGLO + AMP
+ ARABINOSE
-PGLO - AMP
+ ARABINOSE
No glowing
without plasmid
• Identifying Error
• 13. Explain how the following errors could
have happened based on what we know
about the plasmid and AMP
-PGLO +AMP
IN BOTH CASES, THESE
ERRORS COULD HAVE
HAPPENED IF THE
BACTERIA WERE
ACCIDENTALLY GIVEN
THE PLASMID—MAKING
THEM +pGLO- OR- THE
PLATES WERE
MISLABELED
-PGLO - AMP
+ ARABINOSE