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Entry 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 G GATTCC

CCTAAGG CCTAAG 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)

AMPICILLIN RESISTANCE GENE

(Protects against AMP)

GFP GENE

(gene from jellyfish—will glow green if have correct environment/turned on)

THE PLASMID

Gene

Promoter Coding sequenceEnhancer Terminator

• 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

PLASMIDS WITH GFP GENE AND ANTIBIOTIC RESISTANCE GENE

PORES

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 bacteria-so 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

-PGLO -AMP

When no AMP was present the bacteria all survived

• 11. Based on the results of our lab, how do we know the plasmid provides antibiotic resistance?

-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

+PGLO +AMP

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+ ARABINOSE

-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