Redesigning Bacteria:Bacterial Transformation

Student Materials

Introduction...............................................................................................................................2

Pre-Lab Questions and Predictions.............................................................................................4

Lab Protocol...............................................................................................................................5

Data Collection Worksheet........................................................................................................8

Post-Lab Questions....................................................................................................................9

Extension Activity......................................................................................................................11

9/20/19

Redesigning BacteriaIntroduction

It is likely that you have seen or heard of the “Transformers” comics or movies. Transformers are robotic beings that have the ability to change or transform into different forms. Each transformation comes with a new set of amazing characteristics.

The biological world has its own version of transformation. A cell or organism that acquires new DNA also acquires new characteristics and is said to be transformed. Today you are going to transform bacterial cells to produce new cells that are different from what you started with. Bacteria (Figure 1) have genomic DNA in the form of a single double-stranded circular chromosome. The chromosome contains the genes that the bacteria need for normal cellular function. When bacterial cells undergo transformation, naked DNA (DNA without associated proteins) enters the cells from the environment. Often this DNA is in the form of a plasmid, a small piece of double-stranded, circular DNA (Figure 1).

Figure 1. A typical bacterium cell.

Why might the ability to acquire new genetic material be an advantage to a bacterial cell?

If you said acquiring DNA makes a cell “stronger” or “better,” you are right! Plasmids occur naturally and the plasmid DNA encodes a variety of proteins, including proteins for DNA repair or survival in different environments.

Plasmids can also be man-made. One way that a scientist can study a specific gene is to insert the DNA encoding that gene into a plasmid to create a recombinant plasmid and then transform bacterial cells with this new recombinant plasmid. When bacteria replicate the bacterial chromosome, they also replicate the recombinant plasmid DNA. Transformed bacteria divide to make many more cells. In the process, they make many copies of the plasmid and thus the gene being studied. Depending upon the experiment, the bacteria can also express the gene being studied and make huge quantities of protein for research (Figure 2).

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Figure 2. The diagram shows the process of creating a recombinant plasmid by inserting a gene of interest like the gen for human growth hormone into a host plasmid. The recombinant plasmid is then inserted into a bacteria cell.

https://pmgbiology.files.wordpress.com/2014/11/32-recombinant-dna.jpg

Although there are many different types of plasmids, the plasmids that are used in recombinant DNA research must have a few basic components (Figure 3):

Origin of replication (ori) – a site where DNA polymerase and associated proteins attach to start replication.

Multiple cloning site (MCS) – a region that contains multiple different restriction enzymes sites that allow the plasmid to be cut by a variety of different enzymes.

Selectable marker – a gene that produces a protein that allows the transformed bacteria to survive under conditions where untransformed bacteria cannot.

Scientists often use antibiotic resistance genes as selectable markers. A scientist will grow bacterial cells that she has tried to transform on media containing antibiotics. Only cells that have acquired a plasmid will be able to grow. The transformed cells (transformants) have the plasmid, which also contains the gene of interest that was inserted in the multiple cloning site. As these cells multiply, they are, in essence, cellular factories that are able to produce huge quantities of the gene product scientists are studying.

The plasmid used in this lab contains the gene for ampicillin resistance (AmpR

gene). The antibiotic ampicillin inhibits cell wall synthesis so that no new bacterial cells can be made. However, when the cell contains a plasmid carrying the AmpR gene, it can make the enzyme beta lactamase and break down ampicillin before it affects the cells.

Figure 3: A typical recombinant plasmid

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Redesigning BacteriaPre-Lab Questions and Predictions

Directions: After reading through the introduction and protocol for the Redesigning Bacteria lab, answer the questions below.

1. In this lab you will be working with E. coli bacteria. Have you ever heard of E. coli? In what context? How do you think scientists might have modified the bacteria to make sure it is safe to use?

2. What is a plasmid? What advantage or disadvantage do bacteria gain from plasmids?

3. You have learned that bacteria can be easily transformed and that the transformed cells have a trait the untransformed cells do not. Explain why it is more difficult to transform a multi-celled organism to produce an organism with a new trait.

4. As you saw on the starter plate, a single bacterial cell can grow and divide to form a group of many identical cells called a colony. Bacteria can also grow as a “lawn” when the cells are so plentiful that the entire plate is covered with colonies that all grow into each other.

Assume that you successfully transform E. coli in today’s lab. On the plates below draw bacterial growth as colonies or as a lawn, depending on what you expect to see on each type of media. Note: You have millions of cells in each of the tubes. When you add plasmid to a tube only a small percentage of the cells acquire the plasmid.

Examples:

Cells with plasmid on LB

Colonies Lawn

Cells with plasmid on LB with ampicillin

Cells without plasmid on LB

Cells without plasmid on LB with ampicillin

No Bacterial cell growth

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Redesigning BacteriaLab Protocol

You will start with E. coli that are susceptible to ampicillin because they do not have a plasmid with the AmpR gene. The bacteria have been growing on LB agar, which is a gelatinous material that contains nutrients. Since there is no ampicillin in the media the cells can grow and replicate. By carefully following the protocol you will transform some of the bacteria with a plasmid called pUC19. This plasmid contains the AmpR gene

Materials: Check your workstations to make sure all supplies are present before beginning the lab.

Student Workstations: Common Workstation:1 starter plate with E.coli on LB agar 42 C water bath 2 LB/Amp agar plates 37 C incubator (optional) 2 LB agar plates ice bucket with pUC19 plasmid (0.1 µg/µL)

1 tube transformation solution with 1 mL CaCl2

1 tube LB broth with 1 mL broth2 microcentrifuge tubes (2.0 mL)1 p20 micropipette with tips1 p200 micropipette with tips1 p1000 micropipette with tips6 sterile inoculating loops1 ice bucket or Styrofoam cup with crushed ice1 microcentrifuge tube rack2 microcentrifuge tube float1 ultrafine point permanent marker1 waste container

▲Caution: Be sure to keep your reaction tubes on ice through the heat shock in step 10.

Procedure:

1. Use a permanent marker to write your initials on two microcentrifuge tubes. Mark one

microcentrifuge tube + (for plus plasmid) and the other - (for minus plasmid). Place them in the rack on ice.

2. Using a p1000 micropipette add 250 L of transformation solution (CaCl2) to each microcentrifuge tube. Put the tubes back in the ice.

3. Use a sterile loop to gently pick up a single colony of bacteria from your starter plate. The colonies grow on top of the agar—gentle scrape the colony off the top of the agar.

▲Caution: Don’t cut into the agar. Try to remove only a single colony.

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4. Immerse the loop with the colony into the solution at the bottom of the + plasmid tube. Spin the loop between your index finger and thumb until the cells are evenly dispersed in the solution. ▲ Reminder: Put the tube back on ice.

5. ▲Using a new sterile loop, choose another colony and transfer it to the - plasmid tube. Spin the loop to disperse the cells. ▲Reminder: Put the microcentrifuge tubes back on ice.

6. Using a p20 micropipette, add 10 L of the pUC19 plasmid solution to the + plasmid tube. ▲Reminder: Put the microcentrifuge tubes back on ice.

▲ Caution: Do NOT add plasmid to the - plasmid tube. Why not?

7. Incubate both + plasmid and - plasmid tubes on ice for 10 minutes.

8. While the tubes are incubating, locate your two LB/AMP agar plates. Label one LB/AMP plate with your initials and LB/AMP + plasmid as

shown in the diagram to the right. Tip: Label around the bottom (agar side) edge of each plate.

Label the other plate with your initials and LB/AMP - plasmid.

9. Locate your two LB agar plates. Label one LB plate with your initials and LB + plasmid. Label the other plate with your initials and LB - plasmid.

▲ Caution: Confirm with your teacher which plates contain ampicillin before labeling.

10. Carry your ice container over to the hot water bath (42C). Place both microcentrifuge tubes into the warm water for exactly 50 seconds. Immediately return the tubes to the ice container.

▲Caution: For best results the transfer from ice (0C) to 42C and then back to ice must be fast.

11. Incubate both tubes on ice for 2 additional minutes.

12. Remove the rack containing the tubes from the ice and place the rack on your lab bench. Using a p1000 micropipette add 250 L of LB broth to the + plasmid tube and close it.

13. Add 250L of LB broth to the - plasmid tube and close it. ▲Reminder: Use a fresh tip for each transfer.

14. Gently mix the tubes by tapping them with your finger. Incubate both tubes for 10 minutes at room temperature.

15. After the 10 minute incubation, tap the closed tubes with your finger to mix again.

16. Using the p200 micropipette, transfer 100 L of the + plasmid solution onto each of the + plasmid plates.

LB/AMP +plasmid

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17. Using the p200 micropipette, transfer 100 L of the - plasmid solution onto each of the - plasmid plates. Be sure to use a clean tip.

18. ▲Reminder: Use a clean sterile loop for each plate. Spread the cell solutions by holding the flat surface of a loop parallel to the surface of the agar and gently move the loop around the plate.

▲Caution: Use sterile techniques when spreading the bacteria on your plates. o Do not put the sterile loop down on the lab bench before spreading.o The lid of the plate should be held in one hand over the plate while you spread the

suspension with the other hand. o Do not put the lid down on the lab bench!!o Place the used loop into the appropriate trash container. Do not put it down on the lab

bench!

19. Stack your plates and tape them together. Write your name on the tape and place the stack of plates upside down (agar side up) in the 37C incubator until the next day.

20. Clean and disinfect your work area as instructed by your teacher.

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Redesigning BacteriaData Collection Worksheet

Table 1: Bacterial Growth of pUC19 transformants and non-transformants on LB and LB/AMP plates.

Use the circles to indicate where and how much bacteria are growing on the plates. Use the blank spaces for additional observations.

Cells + plasmid Cells - plasmid

LBPlate

LB/AMP Plate

Additional Observations Additional Observations

Additional Observations Additional Observations

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Redesigning BacteriaPost-Lab Questions

Directions: After completing the Redesigning Bacteria lab, answer the questions below.

1. List the plate or plates that represent the controls in this experiment. For each control plate listed, indicate what you expected to see (refer to Pre-lab question 3).

2. Assume that for each control plate listed above you observe a result opposite from what you expected. For example, you observe no bacterial growth when you expected colonies (or a lawn), or you observe lots of colonies when you expected no growth. For each control plate, explain what the unexpected results would mean for your experiment.

3. Look closely at your plates. Were your predictions (see Pre-lab question 3) correct? Explain.

4. How do your results compare to those of your classmates? Provide an explanation for any discrepancies.

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5. You have the following cells:

Cells Cell typeA Untransformed bacterial cells with no plasmidsB Transformed cells that carry a plasmid containing an ampicillin resistant geneC Transformed cells that carry a plasmid containing a penicillin resistant geneD Transformed cells that carry a plasmid containing a tetracycline resistant geneE Transformed cells that carry a plasmid containing both an ampicillin and a

tetracycline resistant genes

You also have the following plates:

Which plate or plates will allow growth of cell type A? Write the letter “A” on each plate where cell type A will grow and form colonies. Repeat this exercise for the remaining cell types B-E.

5. If you and your team were going to try to insert a gene into the pUC19 plasmid, what region of the pUC19 plasmid would you target? Explain your location choice.

6. Describe how scientists can use transformation to produce vast quantities of a protein?

7. Based on what you have learned about plasmids and transformation propose a possible explanation for why so many strains of bacteria are becoming antibiotic resistant.

LB Agar LB Agar w/ tetracycline and ampicillin

LB Agar w/ tetracycline

LB Agar w/ampicillin

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Redesigning BacteriaExtension Activity: Calculating Transformation Efficiency

Transformation efficiency is a quantitative value that describes how effective you were at getting the E.coli bacteria to take up the plasmid DNA and express it. Transformation efficiency is a calculation of the number of transformed cells per microgram (µg) of plasmid DNA used.

Transformation Efficiency = # of transformed cells ÷ amount of plasmid DNA spread on the plate

Calculate Transformation Efficiency

1. You can assume that each colony on your plates grew from a single cell, so the easiest way to determine the number of successfully transformed cells is to count the number of colonies the LB/Amp +plasmid plate.

Number of colonies on LB/Amp +plasmid plate =

2. In order to calculate the amount of plasmid DNA spread on the plate, you must first determine the amount of plasmid DNA you added to the +plasmid tube.

= ×

= µg/µL × µL pUC19 plasmid

3. Because you spread the DNA in the +plasmid tube on multiple plates, you need to calculate the fraction of the DNA that you spread on the LB/Amp +plasmid plate.

= Volume (µL) of cells/DNA spread on LB/Amp +plasmid plate Total volume of cells/DNA in +plasmid tube (µL)

= µL ÷ 510 µL

4. To calculate the amount of plasmid DNA (µg) spread on the LB/Amp +plasmid plate, multiply the amount of plasmid DNA in the +plasmid tube (#2) by the fraction of plasmid DNA used (#3).

= ×

= ×

Concentration of stock plasmid DNA (µg/µL

Volume of plasmid DNA added to

+plasmid tube (µL)

Amount of plasmid DNA (µg) in +plasmid tube Volume of DNA

Amount of plasmid DNA (µg) on LB/Amp

+plasmid plate

Amount of plasmid DNA in +plasmid

tube (µg)

Fraction of cells/DNA spread on LB/Amp +

plasmid plate

Fraction of cells/DNA spread on LB/Amp +

plasmid plate

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5. The transformation efficiency is equal to the number of transformed cells (#1) per microgram (µg) of plasmid DNA spread on the LB/Amp +plasmid plate (#4).

Transformation Efficiency = # of transformed cells ÷ amount of plasmid DNA spread on the plate

Transformation Efficiency = = ÷

Analyze your results.

1. Explain what your transformation efficiency means.

2. The transformation protocol that you used generally has a transformation efficiency of 800-7000 transformants per µg of plasmid DNA. How does your calculation compare with this range?

3. Hypothesize as to what factors might affect transformation efficiency?

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