Summer Research Program for Science Teachers

Justine Kohlmann

Transformation of E. coli with Antibiotic Resistance



In this laboratory exercise, students will introduce a plasmid containing an ampicillin resistance gene into competent E. coli cells. [9-12 Content Standard C- The cell] [Content Standard E- Understandings about science and technology] This exercise will demonstrate how the genetic make-up of an organism can be changed by the introduction of foreign DNA. Once a plasmid containing a gene for antibiotic resistance is introduced using basic transformation techniques, the host cell can express this gene.


Materials

-Competent E. coli cells

-pAMP (plasmids containing the ampicillin resistance gene)

-growth media/ plates

-ampicillin

-pipettes

-ice

-bunsen burners

-37 incubator

-42 and 37 water baths

-test tubes

-ethanol

-cell spreader

-LB broth

[Teaching Standard D- Make accessible science materials]


Procedure:

1. Students receive 2 tubes of competent E. coli cells. pAMP is added to one of the tubes

2. Incubation. Tube containing cells and pAMP is incubated on ice for 20 minutes

3. Preparation of 2 plates with ampicillin:

Plate A for E. coli only

Plate B for E. coli +pAMP

4. Heat shock. Test tube containing cells and pAMP is placed in 42 water bath for 60 seconds and immediately removed

5. Tube is immediately returned to ice for 2-5 minutes

6. LB broth is added to both tubes using sterile techniques

7. Recovery. Test tubes are placed in a 37 shaking water bath for 45 minutes

8. Pipette is used to add normal E. coli cells to plate A and transformed cells to plate B. Cells are spread on plates using a cell spreader that has been dipped in ethanol, flamed, and cooled

9. Plates are placed upside down in incubator (37) overnight


Follow-up:

1. The next day, students observe and count colonies growing on each plate. Students then create a graph to demonstrate results. [Content Standard Unifying Concepts- Evidence, models, and explanation]

2. Students conduct research to answer the following questions:

A.What is happening during the major steps of the transformation: incubation, heat shock, and recovery?

B.How do antibiotic resistant genes work?

3. Students create a poster board that describes the experimental procedure and discusses what is happening in each step of the procedure. They will also demonstrate and explain their experimental results.

 

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