biology 121 lab

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Objectives Genetically transform bacteria with foreign DNA and induce expression of genes encoded on

DNA to produce novel

Isolate chromosomal DNA from

Introduction In this portion of the lab, you will perform a procedure known as genetic transformation. that a gene is a piece of DNA that provides the instructions for making (codes for) a protein. This gives an organism a particular trait. Genetic transformation literally means change caused by genes,

involves the insertion of a gene into an organism in order to change the organism’s trait. transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as

pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a person’s cells with healthy copies of the defective gene that causes the

You will use a procedure to transform bacteria with a gene that codes for Green Fluorescent (GFP). The real-life source of this gene is the bioluminescent jellyfish Aequorea victoria. Fluorescent Protein causes the jellyfish to fluoresce and glow in the dark.

LAB TOPIC 10: Nucleic Acids and Genetic Transformation

Following the procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent

which causes them to glow a brilliant green color under ultraviolet

In this activity, you will learn about the process of moving genes from one organism to another with aid of a plasmid. In nature, bacteria can transfer plasmids back and forth allowing them to share beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to is due to the transmission of

Genetic transformation involves insertion of some new DNA into the E. cells. In addition to one large bacteria often contain one or more circular pieces of DNA called Plasmid DNA usually contains genes for

than one trait. Scientists can use a called genetic engineering to insert

coding for new traits into a plasmid. In case, the pGLO plasmid carries the GFP that codes for the green fluorescent protein and a gene (bla) that codes for a protein that gives the resistance to an antibiotic. The genetically engineered plasmid can then be used to genetically bacteria to give them this new

Figure 10.1 Bacterial cell undergoing genetic transformation with the pGLO plasmid

Exercise 10.1 Bacterial Transformation

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Pre-lab exercises: Since scientific laboratory investigations are designed to get information about a question, our first might be to formulate some questions for this

Can we genetically transform an organism? Which organism is

1. To genetically transform an entire organism, you must insert the new gene into every cell in organism. Which organism is better suited for total genetic transformation, one composed of cells, or one composed of a single

2. Scientists often want to know if the genetically transformed organism can pass its new traits on to offspring and future generations. To get this information, which would be a better candidate for investigation, an organism in which each new generation develops and reproduces quickly, or one does this more

3. Safety is another important consideration in choosing an experimental organism. What traits characteristics should the organism have (or not have) to be sure it will not harm you or

4. Based on the above considerations, which would be the best choice for a genetic transformation: bacterium, earthworm, fish, or mouse? Describe your

The pGLO plasmid also incorporates a special gene regulation system that can be used to expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on transformed cells by adding the sugar arabinose to the cells’ nutrient medium [see appendix for Selection for cells that have been transformed with pGLO DNA is accomplished by growth on

. Transformed cells will express the antibiotic resistance gene and thus will be able to survive in presence of the antibiotic. They will appear white (wild-type phenotype) on plates not arabinose, and fluorescent green when arabinose is included in the nutrient agar

This transformation procedure involves three main steps. These steps are intended to introduce plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly To move the pGLO plasmid DNA through the cell membrane you

Use a transformation solution of CaCl2 (calcium

2. Carry out a procedure referred to as heat

For transformed cells to grow in the presence of ampicillin you

3. Provide them with nutrients and a short

incubation period to begin expressing their acquired

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+ p

G LO

-p G

L O

Transformation Guide

1. Label one of the microfuge tubes containing the transformation solution (CaCl2) with +pGLO and the other -pGLO. Also label them with your group’s name and place them in the foam tube rack.

2. Place the tubes on ice.

3. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Immerse the loop in the transformation solution in the +pGLO tube. Spin the loop between your index finger and thumb until the colony is dispersed (no floating chunks visible). Place the tube back in the rack on the ice. Using a new sterile loop, repeat the process for the -pGLO tube.

4. Examine the pGLO plasmid DNA solution with the UV lamp. Note your observations below. Pipet (or have your instructor pipet) 10 µL of the pGLO plasmid into the +pGLO tube. Close the tube and return it to ice. Do not add plasmid DNA to the -pGLO tube.

5. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so that the tubes stick

+ p

G LO

-p G

L O

ICE

ICE Rack

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6. While the tubes are sitting on ice, label your Four LB Nutrient agar plates on the bottom (not the Lid) as follows:

· Label one LB/amp plate: + pGLO · Label the LB/amp/ara plate: + pGLO · Label the other LB/amp plate: - pGLO · Label the LB plate: -pGLO

7. Heat shock. Using the foam rack as a

holder, transfer both the +pGLO and -pGLO tubes into the water bath, set at 42°C, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottoms of the tubes stick out and make contact with the warm water. When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2 minutes.

8. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 µL of LB nutrient broth to the tube and reclose it. Repeat with a new sterile pipet for the other tube. Incubate the tubes for 10 minutes at room temperature.

9. Tap the closed tubes with your finger to

mix. Using a new sterile pipet for each tube, pipet 100 µL of the transformation and control suspensions onto the appropriate nutrient agar plates.

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10. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR.

11. Stack up your plates and tape them together. Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37°C incubator until the next day.

On which of the plates would you expect

to find bacteria most like the original non-transformed coli colonies you initially observed? Explain your

If there were any genetically transformed bacterial cells, on which plate(s) would they most likely located? Explain your

Which plates should be compared to determine if any genetic transformation has occurred?

What is meant by a control plate? What purpose does a control

5. In the figure below, predict what you would expect to see the

Pre Analysis Questions

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How can we tell if cells have been

Recall that the goal of genetic transformation is to change an organism’s traits Before any change in the phenotype of an organism can be detected, a thorough examination of natural (pre-transformation) phenotype must be made. Look at the colonies of E. coli on starter plates. List all observable traits or characteristics that can be

The following pre-transformation observations of E. coli might provide baseline data to refer to attempting to determine if any genetic transformation has

1. Number of

2. Size of:

a. The largest

b. The smallest

c. The majority of

3. Color of the

4. Distribution of the colonies on the

5. Visible appearance when viewed with

ultraviolet (UV)

6. The ability of the cells to live and

reproduce in the presence of an antibiotic such as

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What happened to the transformed

Observe the results you obtained from the transformation lab under normal room lighting. Then out the lights and hold the ultraviolet light over the

Carefully observe and draw what you see on each of the four plates. Put your drawings in the table in the column on the right. Record your data to allow you to compare observations of the pG LO” cells with your observations for the non-transformed E. coli. Write down the observations for each

How much bacterial growth do you see on each plate, relatively

What color are the

How many bacterial colonies are on each plate (count the spots you

Data Collection: Day 1 – Starter plate Data Collection: Day 2 – Transformation plates

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The goal of data analysis for this investigation is to determine if genetic transformation has

1. Which of the traits that you originally observed for E. coli did not seem to become altered? the space below list these untransformed traits and how you arrived at this analysis for each

Original trait Analysis of

2. Of the E. coli traits you originally noted, which seem now to be significantly different performing the transformation procedure? List those traits below and describe

the changes you

New trait Observed

3. If the genetically transformed cells have acquired the ability to live in the presence of antibiotic ampicillin, then what might be inferred about the other genes on the

plasmid that used in your transformation

4. From the results that you obtained, how could you prove that the changes that occurred were to the procedure that you

Analysis of Results

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Exercise 10.2 Bacterial DNA Extraction

Introduction In this exercise we will be isolating chromosomal DNA from E. coli. [Remember that this is different the plasmid DNA we have examined in the previous section.] To isolate the chromosomal DNA, dodecyl sulfate (SDS), a detergent used in laundry products, removes the lipids from bacterial cell When the cell walls are damaged, the cell lyses, releasing the contents into the bacterial susp ension solution. Along with DNA, enzymes and many other proteins are present in the lysate. Enzymes to DNA must be inactivated at this point by heating the suspension to 60-65°C, a temperature that

proteins but not DNA. DNA must be heated to about 80°C before it denatures. In addition, DNA protected by sodium citrate, which is incorporated into both the bacterial and DNA suspension solutions the kit. The citrate ion is a chelating agent with a strong affinity for magnesium ions. These ions essential to the activity of DNase, the enzyme that degrades The spooling rod is lowered into the lysate. DNA is soluble in water but insoluble in ethanol. As the rod rotated, the DNA fibers come out of solution and attach to the rod. The extracted DNA is then soaked

ethanol to stabilize it and is air-dried to allow the 95% ethanol to

Caution: This kit contains viable, freeze- dried bacteria. The user should wash his/her hands before and after performing the experiment. After completing the experiment, wipe all work surfaces a disinfectant such as 70% ethanol, 4% household bleach, or

Isolation Guide

1. The lab instructor will add 5 mL of the bacterial suspension solution to the freeze-dried E. (in the large glass tube). They will cap the tube tightly and shake it gently, but

the bacterial cells have gone into suspension (this requires about 5 minutes). They then give each student group 2.5 mL of the suspension into a 5 mL

2. Measure out 0.5 mL sodium dodecyl sulfate (SDS) with a pipetter and add it to the E. bacterial suspension. Discard the pipette tips. Cap the tube tightly and invert the tube to avoid excessive bubbling. Repeat this gentle mixing over a 5-minute period. You notice that the suspension becomes more viscous as the bacteria are

3. Stand the tube for 30 minutes in a hot

water bath preheated to 60 to 65°C. While the tubes incubating, place a tube containing 1 mL of 95% alcohol on ice until needed for Step

4. Remove the lysate from the water

bath. Allow the lysate to cool on

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5. Carefully lower the spooling rod (the tip of a Pasteur pipette) into the lysate in the tube. With a pipette, add approximately 1 mL of the cold 95% alcohol solution to spooling tube. To prevent the aqueous and ethanol layers from mixing, tip the tube at a angle, hold the pipette against the side of the tube, and allow the ethanol to flow very down the side of the tube onto the aqueous

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minutes until a visible mass of DNA has attached the

7. Remove the spooling rod and gently

immerse it in new tube of 95% ethanol for 2

8. Remove the rod from the ethanol and

allow the spooled DNA to dry for 5

Figure 1. Hold the spooling rod at an angle of 45° when extracting DNA from the E. coli lysate.

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Appendix: Gene Regulation

Our bodies contain thousands of different proteins that perform many different jobs. Digestive enzymes are proteins; some of the hormone signals that run through our bodies and the antibodies protecting us from disease are proteins. The information for assembling a protein is carried in our DNA. The section of DNA, which contains the code for making a protein, is called a gene. There are over 30,000–100,000 genes in the human genome. Each gene codes for a unique protein: one gene, one protein. The gene that codes for a digestive enzyme in your mouth is different from one that codes for an antibody or the pigment that colors your eyes. Organisms regulate expression of their genes and ultimately the amounts and kinds of proteins present within their cells for a myriad of reasons, including developmental changes, cellular specialization, and adaptation to the environment. Gene regulation not only allows for adaptation to differing conditions, but also prevents wasteful overproduction of unneeded proteins, which would put the organism at a competitive disadvantage. The genes involved in the transport and breakdown (catabolism) of food are good examples of highly regulated genes. For example, the sugar arabinose is both a source of energy and a source of carbon. E. Coli bacteria produce three enzymes (proteins) needed to digest arabinose as a food source. The genes which code for these enzymes are not expressed when arabinose is absent, but they are expressed when arabinose is present in their environment. How is this so? Regulation of the expression of proteins often occurs at the level of transcription from DNA into RNA. This regulation takes place at a very specific location on the DNA template, called a promoter, where RNA polymerase sits down on the DNA and begins transcription of the gene. In bacteria, groups of related genes are often clustered together and transcribed into RNA from one

promoter. These clusters of genes controlled by a single promoter are called operons. The three genes (araB, araA and araD) that code for three digestive enzymes involved in the breakdown of arabinose are clustered together in what is known as the arabinose operon. These three proteins are dependent on initiation of transcription from a single promoter, P . Transcription of these three genes requires the simultaneous presence of the DNA template (promoter and operon), RNA polymerase, a DNA binding protein called araC and arabinose. araC binds to the DNA at the binding site for the RNA polymerase (the beginning of the arabinose operon). When arabinose is present in the environment, bacteria take it up. Once inside, the arabinose interacts directly with araC which is bound to the DNA. The interaction causes araC to change its shape which in turn promotes (actually helps) the binding of RNA polymerase and the three genes araB, A and D, are transcribed. Three enzymes are produced, they break down arabinose, and eventually the arabinose runs out. In the absence of arabinose the araC returns to its original shape and transcription is shut off. The DNA code of the pGLO plasmid has been engineered to incorporate aspects of the arabinose operon. Both the promoter (PBAD) and the araC gene are present. However, the genes, which code for arabinose catabolism, araB, A and D, have been replaced by the single gene which codes for GFP. Therefore, in the presence of arabinose, araC protein promotes the binding of RNA polymerase and GFP is produced. Cells fluoresce brilliant green as they produce more and more GFP. In the absence of arabinose, araC no longer facilitates the binding of RNA polymerase and the GFP gene is not transcribed. When GFP is not made, bacteria colonies will appear to have a wild-type (natural) phenotype—of white colonies with no fluorescence. This is an excellent example of the central molecular framework of biology in action:

DNA→RNA→PROTEIN→TRAIT.

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