What is genetic engineering? How to make glow-in-the-dark bacteria (Traditional method)- Student Handout

Photos by Faith Carter

In this laboratory activity you will introduce bioluminescence genes into E. coli to make the bacteria glow in the dark. This process involves genetic engineering, which is the manipulation of an organism's genetic information by introducing or eliminating specific genes. A more technical term used to describe this process is transformation, which is the uptake of DNA by an organism. Bacterial cells are considered competent when they are capable of uptaking DNA.

You will introduce into E. coli special DNA molecules called plasmids. Plasmids are small circular DNA molecules found in many bacteria. Plasmids can carry one or more genes that encode proteins that confer resistance to certain antibiotics, and scientists can create plasmids carrying genes of interest, such as a glow-in-the-dark gene, even if they are from a different organism than E. coli.

You will first make bacterial cells competent and then introduce plasmid DNA by transformation. One plasmid, pLux, contains the lux genes from Vibrio fischeri., a marine bacteria. V. fischeri produce light and live symbiotically in the light organ of Hawaiian squid. The lux genes are responsible for bioluminescence exhibited by V. fischeri. pLux also contains an antibiotic-resistance gene, ampR. This gene confers ampicillin-resistance in E. coli.

You will examine another plasmid, pUC, by transformation. What genes does pUC contain?

Supplies and Equipment

Per team of four

2 microtubes of competent E. coli in calcium chloride solution.
5 ul 10 ng/ul pUC in a 0.65 ml microcentrifuge tube
5 ul 10 ng/ul pLux in a 0.65 ml microcentrifuge tube
3 ml sterile LB broth in 17 x 100 mm disposable polypropylene tube
2 LB agar + ampicillin plates
Graduate micropipet with tips
2 Sterile 1 ml disposable transfer pipet
2 Sterile 0.3 ml disposable transfer pipet
Container of ice
Permanent marker
Sterile cotton swabs

Per class

Waste containers for biological disposal
37⁰C incubator
37⁰C water bath

Procedure

1. You are provided a microcentrifuge tube containing a calcium chloride (CaCl2) of ice-cold E. coli cells. Label one tube "pLux" and the other tube "pUC". Important: Keep cell solution on ice as much as possible.
   
   
2. Add 5 microliters pUC or pLux plasmid DNA to cells and mix gently. One tube of cells should contain pUC, and the other tube should contain pLux. Allow cells to sit on ice for 10 minutes. A small container of ice works fine for this procedure.
   
   
3. After incubating on ice, heat-shock cells. Bring cells and ice container to water bath. Transfer tube containing cells from ice to 37⁰C. Allow cells to incubate at 37⁰C for 5 minutes, and immediately transfer tube back to ice. Allow cells to sit on ice for 2 minutes.
   
   
4. Using a sterile 1 ml transfer pipet, add 1 ml LB broth to each tube of cells and transfer the tubes to a 37⁰C heat block. Incubate cells for 5-10 minutes at 37⁰C.
   
   
5. Take two LB agar + ampicillin plates. Label the bottom of one plate with "pUC" and the other plate "pLux." Be sure to write your group name on both plates. Put 0.2 ml cells on the correct plate using a sterile 0.3 ml transfer pipet. Then use a sterile cotton swab to spread the liquid evenly on the plate. Properly dispose of the transfer pipet and cotton swab in a waste beaker.
   
   
6. Incubate plates upside down in a 37⁰C incubator overnight.
   
   
7. Remove plates from the incubator and allow to cool to room temperature. Examine the colonies in a very dark room. Which plates have glowing bacteria?
   
   

Questions

1. What is genetic engineering?
   
   
   
   
2. What is a plasmid?
   
   
   
   
3. Draw a diagram of the pLux and pUC plasmids, indicating which genes are present on each plasmid.
   
   
   
   
   
   
4. What is the purpose of the heat shock step during bacterial transformation?
   
   
   
   
5. Why is an antibiotic added to the LB agar plates?
   
   
   
   
6. What does the lux gene encode?
   
   
   
   

BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona October 3, 2001 Designed by: Ken Kubo, Ph.D. Nadja Anderson, Ph.D. nadja@email.arizona.edu   http://biotech.biology.arizona.edu