5 Steps to a 5 AP Biology, 2014-2015 Edition

248 ❯ STEP 4. Review the Knowledge You Need to Score High

Investigation 8: Biotechnology: Bacterial Transformation

This experiment deals with material from Chapter 11, Molecular Genetics. This is the

kind of experiment that can make you feel like a biotech junkie. Here, you use plasmids to

move DNA from one cell to another cell—transformation.You get to play with restric-

tion enzymes, E. coli (Escherichia coli—eww), and gel electrophoresis.

Full understanding of this experiment requires a basic knowledge of

1. What vectors are and how they are made


2. What gel electrophoresis is and how it works


3. What a restriction enzyme is and why it is so important to the field of biotechnology
   You will find all this information waiting for you in Chapter 11. We are not going to cave
   in and explain to you now what those things are. That is something you should do on your
   own.
   OK, we’ll tell you now.... Escherichia coli (usually abbreviated E. coli) is a bacteria
   that is present in everyone’s intestinal tract. It grows in the laboratory as well and contains
   extrachromosomal DNA circles called plasmids.This experiment deals with the process of
   transformation:the uptake of foreign DNA from the surrounding environment. This is
   made possible by the presence of proteins on the surface of cells that snag pieces of DNA
   from around the cell; these DNA pieces are from closely related species.
   The goal of this experiment is to take a bacterial strain that has ampicillin resistance,
   and transfer the gene for this resistance to a strain that dies when exposed to ampicillin.
   After attempting to transform the bacteria, the experimenter can check to see if it was suc-
   cessful by growing the potentially transformed bacteria on a plate containing ampicillin. If
   it grows as if all is well, the transformation has succeeded. If nothing grows, something has
   gone wrong.

Basic Setup

A colony of E. coli is added to each of two test tubes. In one tube a solution is added that

contains a plasmid that carries the ampicillin-resistance gene; the other tube receives no

such plasmid. The waiting game follows, and after 15 minutes on ice, the two tubes are

quickly heated in an effort to shock the cells into taking in the foreign DNA from the plas-

mid. The tubes are returned to ice and the colonies then spread out on an agar plate. They

are sent to the incubator to sleep for the night and grow on the plate.

Results

Four plates are created: two with ampicillin and two without. The bacteria from both test

tubes should happily grow on the plates without ampicillin. The ampicillin-coated plate that

is spread with bacteria from the nontransformed tube is bare—there is, indeed, no growth.

The ampicillin-coated plate that is spread with bacteria from the attempted-transformation

tube shows growth... it may not be the greatest growth ever seen, but it is growth. This

means that some of the E. coli originally susceptible to ampicillin have picked up the resis-

tance gene from the surrounding plasmid and are transformed.

Important point to take from this part of the experiment: “How in the world does trans-

formation work?” Restriction enzymes are added, which cut the DNA at a particular

sequence and open the DNA so that it can be inserted into another such region in the main

E. coli chromosome, which is treated with the samerestriction enzyme. If the opened DNA

from the plasmid happens to find and attach to DNA of the E. coli that is added to the tube,

hallelujah, transformation occurs. In order for this transformation to succeed, the E. coli

must be competent,which means ready to accept the foreign DNA from the environment.

BIG IDEA 3

Genetics and

Information

Transfer

KEY IDEA