Bacterial Transformation LabNotes From the teacher

Day 1: Streaking Starter Plates

Before class: Read the lab. Complete the PreLab for PART A of the lab: o Title and date of the lab o Purpose 1-2 sentences describing the overall goal of the lab; use complete sentenceso Pre-Lab Questions you need to number the question, rewrite the question, and then answer it for

full credit; there are 12 prelab questions and Table 1 that need to be included hereo Lab Procedure Write a procedure for PART A – Streaking the Starter Plates. Use your own

words to describe the steps in experiment in paragraph form.

In class:o Discuss the lab and explain what we will be doing.o Go over the prelab questions to make sure students understand what is going on and WHY. o Streak the starter plates and place them in the incubator overnight.

Day 2: Transformation

Before class: o Read over the procedure for PART B - Transformation of the Lab. o Highlight the Quick Guide pointing out all the important steps. o Cut out the Quick Guide and tape it into your lab notebook. This will serve as the procedure for the

Transformation Portion of your lab.

In class:o Observe the STARTER plates. RECORD A DESCRIPTION of the E. coli at this point in the lab

investigation. Record your observations in your lab notebook. o Carry out the process of TRANSFORMATION (use the Quick Guide). Place your plates in the

incubator.

Day 3: Results

Before class: o Read over the Analysis of Data section of the Lab and revisit/reread the section on transformation

efficiency in the beginning of the lab. o Cut out Table 2 and tape it into your lab notebook.

In class: o Retrieve your plates from the incubator. Work through the Analysis of Data section of the lab and

complete Table 2. o When you are finished with your plates, tape them all completely shut and put them into a Ziplock

bag. The teacher will dispose of your plates. o Answer the Analysis Questions (12) and the write the summary paragraph for homework. Be sure to

include your work for the transformation efficiency portion of the lab.

BACTERIAL TRANSFORMATION LAB

BACKGROUND

In this lab you will perform a procedure known as genetic transformation. Remember that a gene is a piece of DNA which provides the instructions for making a protein. This protein gives an organism a particular trait. Genetic transformation literally means “change caused by genes,” and involves the insertion of a gene into an organism in order to change the organism’s trait. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or spoilage resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with healthy copies of the defective gene that causes the disease.

The concept of cell transformation raises the following questions, among others: To transform an organism to express new genetic information, do you need to insert the new gene into

every cell in a multicellular organism or just one? For laboratory study, which organism is best suited for total genetic transformation — one composed of

many cells or one composed of a single cell? For laboratory study, which would be a better candidate for your investigation — an organism in which

each new generation develops and reproduces quickly or one that does this more slowly? Based on how you answered the first two sets of questions, what organism would be a good choice for

investigating genetic transformation — a bacterium, earthworm, fish, or mouse?

If your answer to the last question is “bacterium,” you are on the right track. Bacteria are singular organisms that reproduce quickly. Genetic transformation of bacteria most often occurs when bacteria take up plasmids from their environment. Plasmids are not part of the main DNA of a bacterium. They are small, circular pieces of DNA that usually contain genes for one or more traits that may be beneficial to survival. Many plasmids contain genes that code for resistance to antibiotics like ampicillin and tetracycline. [Antibiotic-resistant bacteria are responsible for a number of human health concerns, such as methicillin-resistant Staphylococcus aureas (MRSA) infections.] Other plasmids code for an enzyme, toxin, or other protein that gives bacteria with that plasmid some survival advantage.

In nature, bacteria may swap these beneficial plasmids from time to time. This process increases the variation between bacteria — variation that natural selection can act on. In the laboratory, scientists use plasmids to insert “genes of interest” into an organism to change the organism’s phenotype, thus “transforming” the recipient cell. Using restriction enzymes, genes can be cut out of human, animal, or plant DNA and, using plasmids as vectors (carriers of genetic information), inserted into bacteria. If transformation is successful, the recipient bacteria (the recombinant bacteria) will express the newly acquired genetic information in its phenotype (Figure 1).

In nature, the efficiency of transformation can sometimes be low and limited. In the lab, we have discovered several ways to increase the rate of transformation. Now, rather than just a few bacteria taking up a plasmid you want them to use, millions of bacteria can be transformed. The number of bacteria that take up a plasmid successfully is called the “transformation efficiency.” This is one of the values you will calculate in this lab unit.

LEARNING OBJECTIVES

• To demonstrate the universality of DNA and its expression• To explore the concept of phenotype expression in organisms• To explore how genetic information can be transferred from one organism to another• To investigate how horizontal gene transfer is a mechanism by which genetic variation is increased in

organisms• To explore the relationship between environmental factors and gene expression• To investigate the connection between the regulation of gene expression and observed differences between

individuals in a population of organisms

OUR EXPERIMENT

In this lab, we will be using a plasmid called pGLO. We will transform bacteria, E. coli, with a gene that codes for Green Fluorescent Protein (GFP). The real-life source of the GFP gene is the bioluminescent jellyfish Aequorea Victoria, and it causes the jellyfish to fluoresce and glow in the dark. After we transform the bacteria, the bacteria will express their newly acquired jellyfish gene and will produce the fluorescent protein which will allow them to glow a brilliant green color under UV light.

The pGLO plasmid contains the GFP gene and also a gene for resistance to the antibiotic ampicillin. pGLO also incorporates a special gene regulation system that can be used to control the expression of the fluorescent protein in the transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cell’s nutrient medium (by adding sugar to the agar). So, transformed cells (the ones that DO have the pGLO plasmid) will look white on plates not containing the arabinose (sugar), but will glow green on the plates that DO have the arabinose. We are going to transform the bacterial cells with the pGLO plasmid, and then grow the bacteria on several different types of plates to see the differences in gene expression.

The pGLO Plasmid The Specifics:

These are the genes on the pGLO plasmid:1. GFP

The GFP gene encodes for a protein that allows jellyfish to fluoresce at night.

This gene was spliced from a jellyfish genome and inserted into the pGLO plasmid.

Think GFP = green florescent protein. (GLOWING!)2. bla

The bla gene encodes for the enzyme beta-lactamase This enzyme breaks down ampicillin making the E. Coli

resistant to the antibiotic. Think bla = beta-lactamase (RESISTANT TO AMPICILLIN!).

3. araC The araC region is a special gene regulation system (operon),

which can be used to control the expression of other genes. In this case, the araC region is being used to control the GFP gene. In the presence of the sugar arabinose, the gene is switched ON. In the absence of arabinose, the gene is switched OFF. (This is regulated at the level of transcription…more details of what actually happens below…)

i. Regulation of protein expression 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 binds 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.

1. In the presence of arabinose, araC protein promotes the binding of RNA polymerase, and GFP is produced. Cells fluoresce a brilliant green as they produce more and more protein.

2. In the absence of arabinose, araC no longer facilitates the binding of RNA polymerase, and the GFP gene is not transcribed. When the GFP protein is not made, bacteria colonies will appear to have white colonies with no fluorescence.

4. Ori The ori region of the plasmid is the origin of replication for binary fission.

*** NOTE The host E. coli (the bacteria we will use) in this investigation, plasmids, and the subsequent transformants created by their combination are not pathogenic (disease-causing).

Transformation Efficiency

In this investigation, you will also learn how to determine the extent to which the bacteria were genetically transformed. This quantitative measurement is referred to as the transformation efficiency. In many experiments, it is important to genetically transform as many cells as possible. For example, in some types of gene therapy, cells are collected from the patient, transformed in the laboratory, and then put back into the patient. The more cells that are transformed to produce the needed protein, the more likely that the therapy will work. The transformation efficiency is calculated to help scientists determine how well the transformation is working.

When you calculate transformation efficiency, it gives you an indication of how effective you were in getting DNA molecules into bacterial cells. Transformation efficiency is a number. It represents the total number of bacterial cells that express the green protein, divided by the amount of DNA used in the experiment. (It tells us the total number of bacterial cells transformed by one microgram of DNA.) The transformation efficiency is calculated by using the following formula:

Transformation Efficiency = Total number of cells growing on the agar plateAmount of DNA spread on the agar plate (in µg)

Therefore, before you can calculate the efficiency of your transformation, you will need two pieces of information:

1. The total number of green fluorescent colonies growing on your LB/amp/ara plate.2. The total amount of pGLO plasmid DNA in the bacterial cells spread on the LB/amp/ara plate.

---------------------------------------------------------------------------------------------------------------------------------------PRELAB QUESTIONS:

1. In what areas of biotechnology is genetic transformation used? List 3 areas and examples of each. 2. Why are bacterial cells the best candidate for your investigation?3. What is the function of plasmids in bacteria?4. What types of genes do plasmids typically contain?5. What type of bacterial cells are we using for this experiment? 6. What is the plasmid that we will use to transform the bacteria? What genes are found on this plasmid

and what do they do? 7. What is the GFP gene and where is it from? How will this affect the bacteria? How does the GFP gene

get switched on?

8. Normal E. coli is killed by the antibiotic ampicillin. Would you expect bacteria that HAVE NOT been transformed to grow on a plate containing ampicillin? Would you expect bacteria that HAVE been transformed to be able to grow on a plate containing ampicillin?

9. Under what conditions would you expect your E. coli to glow?10. Looking at the quick guide – describe the 4 plates you will be making. What will be in each plate?11. If you shined a black light on the original pGLO plasmid in the vial, would it glow? Why or why not?

(THINK about what it needs to be able to glow!)12. Fill out Table 1. What will we expect to happen in each scenario? (CUT THIS TABLE OUT AND PUT

IT IN YOUR LAB NOTEBOOK)

Table 1. Predictions of Transformation

LB/amp +pGLO LB/amp/ara +pGLO LB/amp -pGLO LB -pGLO

Would you expect the bacteria to grow on these plates? Why or why not?

If bacteria grew, would you expect it to be transformed? Why or why not?

If bacteria grew, would it glow? Why or why not?

---------------------------------------------------------------------------------------------------------------------------------------Part A: Streak Starter Plates

Starter plates are needed to produce bacterial colonies of E. coli on agar plates. Each lab team will need its own starter plate as a source of cells for transformation. Our starter plates are made up of Luria Broth (LB). This is a typical nutrient medium that allows for the growth of bacteria. LB plates should be streaked for single colonies and incubated at 37°C for 24–36 hours before the transformation investigation begins.

Using E. coli and LB agar plates, streak one starter plate for your group in order to generate single colonies from a concentrated suspension of bacteria. A small amount of the bacterial suspension goes a long way. Under favorable conditions, one cell multiples to

become millions of genetically identical cells in just 24 hours. There are millions of individual bacteria in a single millimeter of a bacterial colony.

PROCEDURE – PART A: Streaking Starter Plates1. Obtain a plate labeled LB and write your block #, initials and the word STARTER on the bottom of the

plate (write small).2. Obtain a sterile inoculation loop.3. Streak the plate: (TEACHER WILL DEMO THIS ON THE BOARD!)

a. Insert a sterile inoculation loop straight into the vial of rehydrated bacterial culture. (Try not to touch the sides of the vial.) Remove the loop (You should see a thin film across the loop when you pull it out of the vial.) Streak the plates, as illustrated in Figure 1. Streaking takes place sequentially in four quadrants. The first streak spreads out the cells. Go back and forth with the loop about a dozen times in each of the small areas shown. In subsequent quadrants, the cells become more and more dilute, thus increasing the likelihood of producing single colonies.

b. For subsequent streaks, use as much of the surface area of the plate as possible. After the initial streak, rotate the plate approximately 45 degrees and start the second streak. Do not dip into the rehydrated bacteria a second time! Go into the previous streak about two times and then back and forth as shown for a total of about 10 times.

c. Rotate the plate again and repeat streaking.d. Rotate the plate for the final time and make the final streak.

4. When you are finished your plate, cover it immediately to avoid contamination.5. Place the plates upside down inside the incubator overnight at 37°C or at room temperature for 2–3

days if an incubator is unavailable. Use for transformation within 24–36 hours because bacteria must be actively growing to achieve high transformation efficiency. (Remember, bacterial growth is exponential.) Do not refrigerate before use. This will slow bacterial growth.

6. E. coli forms off-white colonies that are uniformly circular with smooth edges.

---------------------------------------------------------------------------------------------------------------------------------------Part B: Transformation

Now that you have grown your bacteria (E. coli) on the starter plate, it is now time to transform it. We want to put the pGLO plasmid into the bacteria and then see if we can get that bacteria to express its newly acquired genes. We will make 4 plates:

+pGLO on a LB/amp plate WITH the plasmid and the plate has the antibiotic ampicillin+ pGLO on a LB/amp/ara plate WITH the plasmid and the plate has the antibiotic ampicillin and

the sugar arabinose- pGLO on a LB/amp plate NO plasmid and the plate has the antibiotic ampicillin- pGLO on a LB plate NO plasmid and the plate just has the nutrient medium

After we transform the bacteria, they will be plated as described above. The plates will then be put into the incubator to allow for growth, and after the bacteria grow, we will analyze the results.

Before you complete the steps for transformation, write down an observation of what you see on your starter plate.

See the QUICK GUIDE procedure below for directions on how to transform the bacteria.

PROCEDURE – PART B: Transformation QUICK GUIDE!

REMEMBER: Record an observation of your STARTER PLATE before you do your transformation!!

---------------------------------------------------------------------------------------------------------------------------------------Part C: Analysis of Data

Observe the colonies through the bottom of the culture plate. Do NOT open the plates. Count the number of individual colonies; use a Sharpie marker to mark each colony as it is counted. If cell growth is too dense to count individual colonies, record the word "lawn." Complete Table 2 (below) with your data.

Table 2. Results from Transformation LabLB/amp +pGLO LB/amp/ara +pGLO LB/amp -pGLO LB -pGLO

Number of Colonies*

Was the bacteria

transformed?

Did the bacteria glow?

Analysis Questions Copy these questions and write the answers in your lab notebook.

1. Did the pGLO plasmid in the +pGLO vial glow when you shined UV light on it? Why or why not?2. Which plates glowed and why?3. What is transformation efficiency? 4. What is the equation for transformation efficiency? 5. What two pieces of information do you need to calculate transformation efficiency? 6. What plate do you look at to calculate transformation efficiency?7. In order to determine the amount of pGLO plasmid DNA spread on the plate, the equation is:

DNA in µg spread on the plate = (concentration of DNA in µg/µl) x (volume of DNA in µl)

In our experiment it looks like this: ______ = (0.8) x (0.2) [FILL IN THE BLANK TO DETERMINE THE

AMOUNT OF PGLO PLASMID DNA SPREAD ON PLATE! (I am giving you your concentration and volume)]

8. If there were 190 colonies growing on our plate, what would the transformation efficiency be? Show your work and put your answer in a box.

9. Look at your data and the number of colonies on your +pGLO LB/amp/ara plate. Calculate your transformation efficiency for this lab. Show your work and put your answer in a box.

10. [YOU DO NOT NEED TO COPY THIS WHOLE QUESTION – JUST COPY THE PART IN BOLD] Transformation efficiency calculations result in very large, and very small, numbers. For both very large and very small numbers, scientists often use a mathematical shorthand referred to as scientific notation. For example, if the calculated transformation efficiency is 1,000 bacteria/μg of DNA, they often report this as 103 transformants/μg. How would scientists report 10,000 transformants/μg in scientific notation?

11. Report your calculated transformation efficiency in scientific notation. (Convert your answer to Question #9 to scientific notation)

12. Biotechnologists generally agree that the transformation protocol that you have just completed has a transformation efficiency of between 8.0 x 102 and 7.0 x 103 transformants per microgram of DNA. How does your transformation efficiency compare?

Summary Activity In one paragraph, summarize the purpose and results of this experiment. Be specific. What did we find out from completing this experiment?