Microsoft Word - blank.doc

Chapter 20 INVESTIGATIONS

Chapter 20

Protein Synthesis and

Inactivation of Antibiotics

In this investigation, you will examine the effects of ampi-

cillin on two types of bacteria.E. coliMM294/pAmp con-

tains a gene insert that directs the synthesis of a protein that

inactivates ampicillin, whereas E. coliMM294 does not.

Ampicillin inhibits bacterial growth by interfering with cell

wall biosynthesis. Based on your knowledge of protein syn-

thesis, make a prediction about the survival ofE. coli

MM294/pAmp and E. coliMM294 on ampicillin-rich media.

Problem

What effect does the presence of an ampicillin-resistance gene

in a bacterium have on its growth on ampicillin-rich media?

Materials

apron masking tape

safety goggles permanent marker

gloves inoculating loop

10 % bleach Bunsen burner

2 LB agar plates MM294 culture

2 LB ampicillin MM294/pAMP culture

(LB/amp) plates 37 °C incubator

Wear safety goggles at all times.

Wear gloves when performing the experiment.

Disposable latex gloves are best avoided since

allergic reactions to latex have been widely reported.

Disposable polyethylene, PVC, or neoprene gloves

are recommended.

Wipe down all surfaces with 10 % bleach before and

after the laboratory exercise.

All resulting cultures must be immersed in 10 %

bleach before disposal to ensure sterilization.

Do not leave a lit Bunsen burner unattended. Refer

to Appendix C2 for a review of the safe use of a

Bunsen burner.

Wash your hands thoroughly at the end of the

laboratory.

Procedure

1. Put on your safety goggles and gloves, and wipe
   down your bench with a 10 % bleach solution.


2. Obtain two LB plates and two LB/amp plates from
   your teacher.


3. Label the bottom of each plate with your name and
   the date, using a permanent marker.
   4. Label both of the LB plates “amp” for the E. coli
   MM294 cells. Label both of the LB/amp plates “
   amp” for the E. coliMM294/pAMP cells.
   5. Hold your inoculating loop like a pencil and sterilize
   it in the nonluminous flame of the Bunsen burner
   until it becomes red hot. Cool the sterilized loop by
   touching it to the edge of the agar on one of the LB
   plates.
   6. Using the sterilized loop, pick up one colony ofE. coli
   MM294 from a start culture plate. Glide the
   inoculating loop across an LB agar plate, making sure
   not to gouge the agar (Figure 1).

Purpose Design Analysis

Problem Materials Evaluation

Hypothesis Procedure Synthesis

Prediction Evidence

INVESTIGATION 20.1 Report Checklist

Figure 1

Pattern of streaking on an agar plate

7. Resterilize your loop as directed in step 5.


8. Repeat step 6 with E. coliMM294 streaked on an
   LB/amp plate.


9. Resterilize your loop as directed in step 5.


10. Repeat step 6 with E. coliMM294/pAmp streaked on
    the other LB plate.


11. Resterilize your loop as directed in step 5.


12. Repeat step 6 with E. coliMM294/pAmp streaked on
    the other LB/amp plate.


13. Sterilize and cool your inoculating loop.


14. Place all four streaked plates in a stack and tape them
    together. Seal the edges of your plates with masking
    tape.


15. Place the streaked plates upside down in the
    incubator. Alternatively, if you do not have an
    incubator, place the plates in a warm part of the
    room for a couple of days.

NEL Molecular Genetics 695