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Project GENES Laboratory Module

LAB MODULE 2: TRANSFORMATION OF BACTERIA

I. BACKGROUND

INTRODUCTION

Transformation is the process by which foreign DNA is introduced into a bacterial cell in order to “engineer” or change the genetic make-up and phenotype of bacteria. In this lab we will be using a recombinant DNA plasmid that contains two important genes. The first is a gene that causes the bacteria to become resistant to the antibiotic ampicillin (Ampr). The second, is a gene from Jellyfish called Green Fluorescent Protein (GFP) which makes Jellyfish glow green when activated by blue light.

The goal of today’s lab is to get this recombinant DNA plasmid inside of bacteria, so that the genes on the plasmid (Ampr and GFP) are expressed (transcribed and translated so that their protein products are made). DNA molecules are quite large and negatively charged, so normally they would be excluded from crossing the bacteria’s plasma membrane. To combat this bacteria can be treated with CaCl2 which makes them “competent” to take up DNA. If we incubate competent bacteria with DNA, and then give the cells a sudden heat shock, a small percentage of the cells will take up the foreign DNA plasmid. By plating the cells on agar plates containing the antibiotic ampicillin, we can select for only the cells that took up the DNA. Cells that did not take up the DNA are unable to grow in the presence of ampicillin, while those that took up the plasmid produce a protein (enzyme) called β-lactamase which breaks down the ampicillin, so that the bacteria can grow.

The results are bacterial colonies containing the recombinant plasmid that are capable of producing β-lactamase and GFP. If we are successful, you will have created bacteria that have been transformed with the DNA plasmid and that glow bright green!

CAREERS IN DNA SCIENCE RELATED TO THIS LAB

Genetic engineering research scientists, or biotechnologists, manipulate and modify the genes, or hereditary makeup, of microorganisms, plants, and animals. They are specialists in the field of genetics and conduct research in a broad range of biological sciences including biochemistry, botany, embryology, and microbiology. They have developed techniques with numerous important applications in the fields of medicine, agriculture, and animal husbandry.

Career Spotlight:

Harvard U. Researchers Transform Bacteria: Into A Biofuel Powerhouse. Ctrl + Click on image for the link to this article.

YOUR PRE-LAB E-PORTFOLIO ASSIGNMENT

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

  1. What can we use DNA for?
  1. Why would we want to put DNA into bacteria?
  1. What is a plasmid?
  1. What is ampicillin?
  1. Check out this YouTube video link that talks about the role of DNA plasmids in Genetic engineering.

Video

II. OBJECTIVES OF THIS LABORATORY ACTIVITY

●Enable students to observe the experimental process called bacterial transformation.

●Enable students to observe the change in phenotype caused by the uptake and expression of a known plasmid sequence

●To understand the importance of a marker gene in molecular biology.

●To become familiar with the sterile techniques that are used to handle bacteria.

III. LABORATORY PROCEDURE

Step 1: Transformation of E. coli with pGreen.

  1. Obtain 2 of the 1.5 ml tubes.
  2. Mark ‘+ plasmid’ on one and ‘- plasmid’ on the other
  3. Add 10ul of the pGREEN DNA plasmid to your + plasmid tube
  4. Add 250ul of calcium chloride to each tube.
  5. Add one loop full of MM294 E.coli cells to each tube (use sterile technique).
  6. Keep tubes on ice for 20 min.
  1. While the tubes are incubating, obtain 2 LB-Amp and 2 LB plates and label on the bottom as follows:
  1. After 20 min on ice, heat shock the bacteria cells by going from ice directly to heat bath for 45 seconds
  2. Gently agitate while in heat bath
  3. Go from heat bath directly back to the ice for 2 minutes
  4. Pipette 250ul of broth (LB) into each tube and gently tap to mix.
  5. Place tubes in rack at room temperature for 20 min.

Step 2: Plate the E.coli on plates of LB agar.

  1. Remove cells from tubes and put on your labeled plates.
  2. Do one plate at a time by transferring 200ul from each tube to the correct plate.
  3. Spread as demonstrated by your instructor.
  4. Repeat with other plates
  5. Stack plates on your table and your instructor will incubate the plates at 37°C overnight.
  6. Observe and record your results the next day.

Record your observed results in the chart below. If there are too many colonies to count just record LAWN.

Plate Designation / Number of Colonies / Color of Colonies

Feel free to add pictures of your results to your ePortfolio.

IV. POST-LAB E-PORTFOLIO ASSIGNMENT

Please follow the link below to answer your post-lab questions. Make sure that you have read and completed the activities for the entire module before you begin. Ctrl+Click to follow the link.