Bacterial Transformation

For your lab report you will be using the ampR plasmid from the virtual lab. The procedures that I included are for a face-to-face lab that uses a different plasmid, but the steps are the same. This was included so you can get more detail about the actual steps involved in bacterial transformation. In your report use the ampR plasmid.

* Introduction
* Hypothesis
* Think about what the gene in the added plasmid does for the bacteria…
* Materials (DO NOT tell me you need a computer, I listed them for you)
* E. coli bacteria
* ampR plasmid
* CaCl2 solution
* Agar plates labled:
* – ampR, – Amp (amicillin) (this means you will add bacteria WITHOUT the additional ampR plasmid to a plate WITH NO ampicillin in the growth medium)
* – ampR, + Amp (this means you will add bacteria WITHOUT the additional ampR plasmid to a plate WITH ampicillin in the growth medium)
* + ampR, – Amp
* + ampR, + Amp
* Hot water bath (for heat shocking the bacteria)
* Ice bath
* Transfer loop for transfering the bacteria from solution to the agar plates
* Incubator
* Procedures
* I would refer to the hands on lab protocol I included below as a reference.
* Please note it uses a different plasmid and one plate has a different medium added to it but otherwise it is the same.
* Data Table
* Observations of the bacterial plates made during the analysis.
* There are four DIFFERENT plates, two different plates without the plasmid added and two plates with the plasmid added.
* Conculsion
* Summarize your results.
* Discuss if your hypothesis is supported or not and how you know.

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.
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.

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.

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.
Sterile Laboratory 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.
ALWAYS NEVER
Always wash your hands and work surface before beginning.
Never have food on your work surface.

Always keep the lid of the petri dish on it or over it at all times. Microbes are everywhere!
Never lay the lid of the petri dish or culture tube on the lab bench.

Always open all sterile tools carefully.
Never touch the end of a tool that touches bacteria.

Always keep hair pulled back and use goggles when flame is present.
Never throw biohazard materials in the regular trash.

Always wash your hands thoroughly with soap and hot water before leaving the lab.

Now you’re ready to begin the experiment.

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.

Key Concepts II: Electrophoresis
In the 1960s, scientists discovered that bacteria have enzymes that cut, or “digest,” the DNA of foreign organisms and thereby protect the cells from invaders such as viruses. Scientists have now isolated several hundred of these enzymes, known as restriction enzymes, or restriction endonucleases. Each is able to recognize and cut at a specific DNA sequence, known as a recognition sequence.

The discovery of restriction enzymes made genetic engineering possible because researchers could use them to cut DNA into fragments that could be analyzed and used in a variety of procedures.
In this part of the laboratory, you will use gel electrophoresis to separate samples of DNA that have been digested by restriction enzymes. Then you will compare fragments of unknown size to fragments of a known size to calculate the unknown fragment sizes.
Let’s begin by looking at how restriction enzymes work.

How Do Restriction Enzymes Work?
Like all enzymes, restriction enzymes are highly specific. They cut DNA only within very precise recognition sequences. Study the illustrations below to see three different recognition sequences. The red line shows where the enzymes will cut the DNA. Notice that all of these recognition sites are symmetrical, or what is called “palindromic.” This means that the recognition sequence on one DNA strand reads in the opposite direction on the complementary strand.
Cut
Cut
Cut

Next let’s look at the laboratory procedures for cutting and separating DNA fragments.

Cutting DNA with Restriction Enzymes
Microscale Quantities
We use very small quantities when working with DNA, so the volumes and tools are adapted to this microscale. In the metric system, the prefix “micro-” indicates one-millionth. It is symbolized by μ, the Greek letter mu. Some examples are:
1 ml = 1000 µl (1000 microlitres)
1 mg = 1000 µg (1000 micrograms)
You begin by mixing DNA with one or more restriction enzymes in a small plastic microcentrifuge tube. The total volume of the mix is about 20 μl.

Most restriction enzyme reactions are incubated at 37°C for one hour. After incubation, you can analyze the DNA or use it in other kinds of reactions, such as the bacterial transformation you did in the first part of this lab.
In the next procedure, you will see how to analyze separate DNA fragments with gel electrophoresis.

Gel Electrophoresis
Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field. The direction of movement is affected by the charge of the molecules, and the rate of movement is affected by their size and shape, the density of the gel, and the strength of the electrical field.
DNA is a negatively charged molecule, so it will move toward the positive pole of the gel when a current is applied. When DNA has been cut by restriction enzymes, the different-sized fragments will migrate at different rates. Because the smallest fragments move the most quickly, they will migrate the farthest during the time the current is on. Keep in mind that the length of each fragment is measured in number of DNA base pairs.
In your laboratory, you will prepare and “run” your own gel electrophoresis.

Design of the Experiment II
In your laboratory you will be given three samples of DNA obtained from a virus, the bacteriophage lambda. One sample will be uncut DNA, one will be incubated with the restriction enzyme HindIII, and one will be incubated with EcoRI. You will separate the fragments of DNA by electrophoresis, stain the DNA for visualization, and determine the fragment sizes formed in the EcoRI digest.
The figure below is an overview of the procedure, using generalized DNA samples. Over the following several pages we look at the procedure step by step.