AP Bio Lab 8: Transformation We will start on TUESDAY! All the following information can be found at: http://phschool.com/science/biology_place/labbench/lab6/intro.html Homework: Review all the information on this website, and complete self-quizzes for understanding of lab procedure and results.

Key Concepts I: Bacterial Transformation Genetic transformation occurs when a host organism takes in foreign DNA and expresses the foreign gene. In this part of the lab, you will introduce a gene for resistance to the antibiotic ampicillin into a bacterial strain that is killed by ampicillin. If the susceptible bacteria incorporate the foreign DNA, they will become ampicillin resistant.

Bacterial Colonies The bacterium you use in your laboratory activity is Escherichia coli, which has been grown in a petri dish on Luria Broth (LB) agar. Each colony in the petri dish is made up of millions of individual cells.

E. coli Bacteria Escherichia coli is the most common bacterium in the human gut. It has been extensively studied in the laboratory and is an important research organism for molecular biology. E. coli reproduce very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight. Like all bacteria, E. coli has no nuclear envelope surrounding the bacterial chromosome and thus no true nucleus. All of the genes required for basic survival and reproduction are found in the single chromosome. Some E. coli cells also contain plasmids, small DNA molecules that carry genes for certain specialized functions, including resistance to specific drugs.

Plasmids Plasmids are circular pieces of DNA that exist outside the main bacterial chromosome and carry their own genes for specialized functions. In genetic engineering, plasmids are one means used to introduce foreign genes into a bacterial cell. To understand how this might work, consider the plasmid below.

Plasmids cont. Some plasmids have the ampR gene, which confers resistance to the antibiotic ampicillin. E. coli cells containing this plasmid, termed "+ampR" cells, can survive and form colonies on LB agar that has been supplemented with ampicillin. In contrast, cells lacking the ampR plasmid, termed "–ampR" cells, are sensitive to the antibiotic, which kills them. An ampicillin-sensitive cell (–ampR) can be transformed to an ampicillin-resistant (+ampR) cell by its uptake of a foreign plasmid containing the ampR gene. To transform cells, you first need to make them competent to take up extracellular DNA.

Recombinant DNA

Competent Cells E. coli cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. Such cells are said to be "competent." Cells are made competent by a process that uses calcium chloride and heat shock. Cells that are undergoing very rapid growth are made competent more easily than cells in other stages of growth. The growth rate of a bacterial culture is not constant. In the early hours (lag phase), growth is very slow because the starting number of dividing cells is small. This is followed by a time of rapid cell division known as the log phase. The actual length of each phase depends on the temperature at which the cells are incubated. In this lab, you will start with cells that should be in the log phase.

Design of the Experiment I You now have an understanding of how cells are prepared for transformation. Before beginning the experiment, it's important to review the basics of sterile procedure. The techniques of sterile procedure apply to any activity in which you work with bacteria or fungi. Since you are working with E. coli bacteria in this laboratory, it is important that you not contaminate your work with any foreign bacteria or expose yourself to potentially hazardous bacteria. The chart below summarizes the basics of sterile procedure.

Go to website http://phschool.com/science/biology_place/labbench/lab6/design1.html

Transformation Procedure In your laboratory, you use plasmids that carry the ampR gene to transform E. coli cells that lack this gene. The illustration below outlines the major steps in this procedure. You also prepare a second group of E. coli cells as a control to verify that E. coli will not grow on agar with ampicillin unless it is transformed, and that nothing in the procedure itself affects the survival of E. coli. The procedure is the same for both groups of cells except in step 2, where you add ampR plasmids to the experimental cells but not to the control cells.

Closer Look: Transformation Procedure: Step 1

Closer Look: Transformation Procedure: Step 2

Closer Look: Transformation Procedure: Step 3

Closer Look: Transformation Procedure: Step 4

Closer Look: Transformation Procedure: Step 5 The cells are incubated for 24 hours

Closer Look: Transformation Procedure: Step 6

Analysis of Results I If there is no ampicillin in the agar, E. coli will cover the plate with so many cells it is called a "lawn" of cells. Only transformed cells can grow on agar with ampicillin. Since only some of the cells exposed to the ampR plasmids will actually take them in, only some cells will be transformed. Thus you will see only individual colonies on the plate. If none of the sensitive E. coli cells have been transformed, nothing will grow on the agar with ampicillin.

Analysis of Results I

Homework  due tomorrow Review Transformation Process on this website, and complete self-quizzes for understanding of lab procedure and results. http://phschool.com/science/biology_place/labbench/lab6/trananim.htm l

Tuesday  Start Transformation Lab Complete DNA transformation, set up plates Homework  answer Analyzing Results Questions ….

Wednesday  gather lab data Gather & Record data Answer Analyzing Results Questions 1-5 on pg. S106 in lab booklet Calculate Transformation Efficiency, pg.S106-S108 Homework  Complete Evaluating Results Questions 1-5 on pg.S109 in lab booklet