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Bacterial Transformation with “glowing” DNA (GFP on a plasmid)

Bacterial Transformation begins with generating a recombinant DNA consisting of a plasmid (small circular DNA molecule distinct from the chromosomal DNA) with an inserted piece of DNA, the gene of interest (in this case GFP). Investigators at the University of Arizona selected a plasmid named pBAD. The p signifies it is a plasmid and BAD is a designation due to the presence of a promoter called BAD. This plasmid is considered to be an expression vector, the BAD promoter will be responsible for the GFP expression. If a piece of DNA with a gene of interest is placed into one of the restriction enzyme sites in pBAD’s multiple cloning site (MCS) and sealed with DNA ligase a recombinant plasmid has been created. If this recombinant plasmid is placed in a bacterial cell the gene can be expressed- the mRNA will be produced and this mRNA will be translated by a Ribosome to generate a protein. The gene which was put into the pBAD plasmid is from a jellyfish and produces a protein that fluoresces under ultraviolet (uv) light. The plasmid is called pBAD-gfpuv.

Some questions to get you thinking about today’s lab:

What is a plasmid?

What is ampicillin?

Materials for each group (students should work in groups of 4):

Tube of plasmid DNA (tube labeled GFP) 1 tube of LB broth

One tube of E. coli bacteria (tubes on ice) micropipette

2 LB agar plate micropipette tips

2 LB agar plates with selection (ampicillin) (1 black stripe) transfer pipette

Q-tips or inoculation loops

Materials to share:

Water bath at 42°C

Ice

Trash containers/biohazard waste bag

UV lights

Protocol:

1. Pipette the 20 μl of DNA solution from your GFP DNA tube into your E. coli bacteria tube and label the tube with your initials. As a control you will not add DNA to an E. coli bacteria tube, label it and process it in the same way you do for the GFP DNA transformation tube.

2. Put tubes on ice for 5 minutes. Why do you think we put the tubes on ice?

3. In the meantime, each group should get two LB agar plate and two LB agar + ampicillin plate (with one stripe). Write your initials and your section number on your plates. You will be plating bacteria with DNA on both plates. Where is the best place to label your plates? What is the control you are conducting?

4. Put tubes directly from ice into 42°C water bath for 50 seconds. What do you think heating the tubes does?

5. Put tubes directly from water bath onto ice for 2 minutes.

6. Add all of one tube of LB broth into the transformation tube. This can be done by pipetting the entire volume which should be 500ml or if the correct sized pipettes are not available, tipping the LB tube upside down onto the transformation tube and flicking the LB tube contents into the transformation tube. Do the same with your control tube. Incubate at room temperature or in your hand for 10 minutes. What is the LB broth for? Why would you want the cells to be as warm as your hand?

7. After the 10 minute incubation, add an equal amount of LB Broth, DNA and E coli. from your transformation tube onto one of your LB agar plate and LB agar + ampicillin plate by pipetting an equal number of onto each plate. This can be done by using a transfer pipet and adding an equal number of drops to each plate or by pipetting half the volume with an adjustable pipet (the volume in the tube is approximately 550ml, half would be 275ml). Do the same with your control.

8. Spread the solutions on the plates using a sterile Q-tip (spread LB agar plate first and then LB agar + ampicillin plate with one stripe--Why do you think you want to spread the solutions onto the plates in this order?). Be careful not to stab the agar.

9. Put your plates in a 37°C incubator for 24 hours. Why 37°C?

10. What do you expect to grow on each of the plates?

| | | | |

| |LB agar |LB agar + ampicillin |Do you expect to see any difference in|

| | |(1 black stripes) |bacterial growth on the plates? |

| | | | |

|E. coli + GFP DNA | | | |

| | | | |

|E. coli control (no| | | |

|DNA) | | | |

Day 2:

What do you see on your plates?

Now look at your plates with UV light. What do you see?

Fill in the table with your data - What do you see on each type of plate?

| | | |

|Type of agar plate |LB agar |LB agar + ampicillin |

| | |(1 black stripes) |

|What kind of growth and | | |

|phenotype(s) did you see | | |

|with DNA? | | |

|What does this tell you | | |

|about the DNA? | | |

|What kind of growth and | | |

|phenotype(s) did you see | | |

|with no DNA? | | |

|What does this tell you | | |

|about the DNA? | | |

What does the DNA allow the bacteria to do?

You added a gene from jellyfish that causes the jellyfish to glow. When you looked at the cells with UV light did your bacteria glow? Explain your results.

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