AP Biology 12 – Lab 6A – Transformation 1

AP Biology 12

AP Lab 6 – Molecular Biology

6A: Bacterial Transformation (Adapted Lab)

Glowing Bacteria: Transformation of a FireFly Gene

***The following lab uses a procedure adapted from Ward’s Natural Science Establishment (2002). The

procedure has been adapted to allign with the Advanced Placement Program Lab Manual Learning Objectives

(2001). The objectives, materials, procedure, analysis, and discussion questions are a combination from both

lab resources.***

Overview

Plasmids containing specific fragments of foreign DNA will be used to transform Escheruchia coli cells, passing

on antibiotic (ampicillin resistance) and foreign gene (firefly luc) expression.

Objectives

1) To understand the principles of bacterial transformation, including how plasmid vectors are engineered and taken up by cells.

2) To understand how antibiotic resistance can be transferred between cells. 3) To understand how a foreign gene (firefly luc) can be transferred between cells. 4) To compare multiple experimental controls. 5) To determine the factors that affect transformation efficiency. 6) To calculate transformation efficiency.

To answer: What are your predictions in terms of colony growth and luminescence?

Introduction

Please read the background information provided by Ward’s Natural Science Establishment (2002) in the

following pages. The materals, procedure, results, analysis, and discussion questions follow after the

background information.

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Materials

Day 1 Per group: Media/solutions: 1 E. coli on Luria agar slant 2 Luria agar plate with ampicillin (LB/Amp) 2 Luria agar plates (LB)

0.5 mL Calcium chloride solution 10 μl Plasmid pBestLuc solution 0.5 mL Luria broth

Equipment:

2 microcentrifuge tubes 4 sterile 1 mL pipettes

1 pipettor 2 sterile inoculating loops 1 capillary micropipette with plunger 4 Nitrocellulose membranes 1 plastic forceps in ethanol (for membranes) 1 timer 1 marking pen 1 Ice bath 1 Hot water bath with thermometer (ready to 42°C)

For the class: 1 +37°C incubator Day 2

Per group: Media/solutions:

Prepared plates from Day 1: LB/Amp+ LB/Amp- LB

LB- 2.0 mL Luciferin

Equipment: 1 sterile pipette 1 Pipettor 1 Plastic forceps in ethanol

Per class:

1 Room blacked out (W203A)

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Procedure

You will insert the plasmid pBestLuc into competent E. coli cells. Before coming to the lab, be sure to

complete all the predictions.

Note: Prior to conducting the lab, make sure all materials are present and ready to use. A 42°C water bath

should be available. The calcium chloride and plasmid pBestLuc solutions should be in an ice bath and kept

cold throughout the experiment. Note that all volumes must be absolutely precise. There will be no waste for

any of the solutions/broths. If you use too much, you will run out. Also note the sterile equipment.

Contamination is a risk, so you must follow the procedure with diligence. Ms. Wood will be placing you into

three lab teams. Before starting the lab, you will have a team meeting to compare your pre-labs and decide on

“jobs.” Ensure you have completed all predictions.

Day 1

1) Mark one sterile centrifuge tube “+”: this tube will have plasmid added to it. Mark the other 1 centrifuge tube “-“: this tube will have no plasmid added.

2) Using a sterile 1 mL pipette, add 0.25 mL (250μL) of ice-cold calcium chloride solution to each tube.

3) Obtain the Luria agar slant with actively growing E. coli. Using a sterile inoculating loop, remove a loopful (3mm) of the E. coli from the surface of the agar. Gently move the loop across the surface of the agar to collect some of the bacteria. Be careful not to remove any agar with the loop.

4) To remove the bacteria from the inoculating loop, place the loop into the calcium chloride and twirl rapidly. Dispose of the loop into the appropriate waste container. Gently tapping the loop against the side of the tube may also help dislodge the bacteria.

5) With a new inoculating loop, repeat steps 3 – 4 for the other tube.

6) Use the capillary micropipette and plunger to add 10 μL of the plasmid BestLuc solution to the (+) tube.

7) Gently tap both tubes with your finger to mix the solutions.

8) Keep both tubes on ice for 15 minutes.

9) While the tubes are on ice, obtain 2 LB agar plates and 2 LB/Amp agar plates. Label each plate on the bottom as follows: “LB+”, “LB-“, “LB/Amp+”, and “LB/Amp-.” Also label each plate with your team name.

10) Using forceps (that were soaked in ethanol) place a piece of nitrocellulose membrane on the surface of each of the agar plates. Be sure to replace the lid as soon as possible to minimize contamination. Be very careful when handing the nitrocellulose membrane; it is very fragile.

11) The E. coli bacterial cells must be heat-shocked to allow the plasmid to enter. Remove both tubes from the ice and immediately place them in a 42°C water bath for 75-90 seconds. It is essential that the cells be given a sharp and distinct shock, so take the tubes directly from the ice bath to the 42°C water bath.

12) Remove the tubes from the 42°C water bath and immediately return the tubes to the ice bath. Keep the tubes on ice for 2 minutes.

13) Remove the tubes from the ice bath and add 0.25 mL (250μL) of room temperature Luria broth to each tube, using a sterile 1 mL pipette. Gently tap the tubes with your finger to mix the solution. The tubes may now be kept at room temperature. Any transformed E. coli cells are now resistant to ampicillin.

14) Using a different sterile 1 mL pipette (the third one now), add 0.20 mL (200μL) of the solution from the (+) tube to the centre of the LB/Amp+ plate, on top of the nitrocellulose membrane. Gently rock the plate to ensure the solution thoroughly covers the surface of the nitrocellulose membrane. Immediately cover with the lid.

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15) Repeat step 14 with the same 1mL pipette for the LB+ plate.

16) Using a different sterile 1 mL pipette (the forth one now), repeat step 14 for the (-) tube and both the LB/Amp- and LB- plates.

17) Allow plates to sit undisturbed for one hour. After one hour, place the plates in a 37°C incubator, inverted, overnight.

Day 2

1) Remove your plates from the incubator.

2) Count the number of colonies growing on each plate and record the value in the results; use a permanent marker to mark each colony on the bottom of the plate (not the lid) as it is counted. If the cell growth is too dense to count individual colonies, record “lawn.” Do not open the plates for counting!

3) Remove the lids of the plates and place the lids upside down on a flat surface. Make sure there is a small label on the outside edge of the lid to indicate the culture.

4) Add 0.5 mL of 1 mM luciferin solution to the centre of each lid.

5) Using the forceps, carefully remove the nitrocellulose membrane containing the transformed colonies of E. coli from your Luria agar plates and place in the lid of the plate on top of the luciferin solution.

6) Take into a dark area (W203A) and observe any luminescence.

7) Complete the questions in the analysis of results section.

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Results Table 1. Number of colonies of E. coli after overnight incubation.

Plate Number of colonies after overnight incubation.

Prediction Actual

LB/Amp+

LB/Amp-

LB+

LB-

Table 2. Observations of luminescence of E. coli after overnight incubation and exposure to 1 mM luciferin solution.

Plate Luminescence? All colonies, some colonies, or no colonies

Prediction Actual

LB/Amp+

LB/Amp-

LB+

LB-

Other Observations and Experimental Notes:

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Analysis of Results

1. Compare and contrast the number of colonies in each of the following pairs of plates. What does each pair of results tell you about the experiment?

a. LB+ and LB-

b. LB/Amp- and LB/Amp+

c. LB/Amp+ and LB+

2. Compare and contrast the luminescence in each of the following pairs of plates. What does each pair of results tell you about the experiment?

a. LB+ and LB-

b. LB/Amp+ and LB+

c. LB/Amp+ and LB-

3. Calculate the transformation efficiency (number of colonies per μg of plasmid) using the steps below. Use the data from the LB/Amp+ plate. Concentration is in μg/μL; mass in μg; volume in μL

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Discussion Questions

1. Based on your observations, did transformation occur? Why or why not?

2. What was the transformation efficiency of plasmid pBestLuc? What factors might influence transformation efficiency? Explain the effect of each that you mention.

3. Explain the importance of utilizing experimental controls. There are two types of controls: a positive control and a negative control. Using the internet, research both types of controls and describe them. (Cite your source!) There were four plates cultured in this lab. Determine which plate was the experimental test and whether the other three were positive or negative controls. Explain your reasoning.

Conclusion

Refer to the criteria on the lab rubric.

(To make sure you’ve understood the concepts of this experiment, answer questions #5-8 on page 334 in Hebden.)