There are two locations of DNA in a bacterial cell. Chromosomal DNA in a bacterial cell carries genes needed for the hereditary characteristics that are essential for bacterial growth and reproduction. Plasmids are small pieces of circular DNA that are separate from the chromosome and replicate independently. Bacteria naturally have one or more plasmids with genes for one or more traits that may be beneficial, but not essential, to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The recent occurrence of bacterial resistance to antibiotics is due to the transmission of plasmids.

A gene is a segment of DNA which “codes for” or provides the instructions for making a protein. This protein gives an organism a trait. Genes can be transferred from one organism to another by transformation which involves the insertion of the gene into an organism to change the organism’s trait.

DNA –> RNA –> Protein –> Trait

The transfer of genes by the transformation process 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 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 GFP gene in this exercise was originally found in the bioluminescent jellyfish Aequorea Victoria. The GFP gene codes for green fluorescent protein which allows the jellyfish to fluoresce and glow in the dark.

This procedure transforms E. coli bacteria by introducing a unique, genetically engineered pGLO plasmid in which three genes have been inserted. Following the transformation procedure, the competent bacteria cells (able to take up the plasmid) will express their newly acquired genes. Most bacterial cells are not competent; however, mixing the cells with calcium chloride during their growth phase will induce competence by the interactions of calcium ions with the cell envelope and the DNA phosphate backbone.

The three genes inserted on the pGLO plasmid are listed in the table. Each gene codes for a specific protein which will enable the E. coli to demonstrate different traits.

pGLO incorporates an inducible operon which controls the expression of the Fluorescent Protein in transformed cells. The BLA gene and Ara-C gene are continually transcribed and translated into protein. The gene for GFP, on the other hand, is regulated by the Repressor protein and will not be transcribed without the addition the sugar arabinose to the cells’ nutrient medium. The arabinose will inactivate the repressor protein thus allowing the GFP gene to be transcribed.

Selection for E. coli that are transformed with pGLO is accomplished by growth on nutrient agar containing the beta-lactam antibiotic, ampicillin, and on nutrient agar containing ampicillin plus the sugar, arabinose. Transformed cells will demonstrate the traits of the additional genes. The E. coli that were not transformed will be killed by the antibiotic, ampicillin.

Pre-Assessment

PROCEDURE

Transformation Procedure

1.  Your instructor labeled the lid of one microcentrifuge tube P (pGLO plasmid + E. coli) and the other lid C for control (only E. coli).

2.  Your instructor pipetted 5µL pGLO plasmid into the microcentrifuge tube marked P and 50µL of competent E. coli into both the C and P microcentrifuge tubes.

3.  Your instructor placed both microcentrifuge tubes in the ice chest. The microcentrifuge tubes must be on ice for 20 minutes before you continue the experiment.

4.  After the microcentrifuge tubes have been on ice for 20 minutes, transfer the microcentrifuge tubes to a floater in the 37^oC water bath for EXACTLY 3 minutes. During this time, the competent E. coli are taking up the pGLO plasmid. The heat shock alters membrane fluidity creating pores and allows for pGLO plasmid to enter the bacterial cells.

5.  Immediately transfer the microcentrifuge tubes back into the ice chest for one minute.

6.  Add 0.5mL Luria broth to the P microcentrifuge tube. Add 0.5mL Luria broth to the C microcentrifuge tube. Incubate the microcentrifuge tubes in a floater in the 37^oC water bath for at least 30 minutes. The transformed E. coli use the nutrients and time to transcribe and translate genes.

7.  Pick up from the side counter:

1 Luria agar plate (there will be no lines on the side of the Luria agar plate to designate nothing was added to the Luria agar)

2 Luria plus ampicillin plates (there will be one line on the side of the Luria agar plate to designate one item was added to the Luria agar)

1 Luria plus ampicillin plus arabinose plate (there will be two lines on the side of the Petri plate to designate two items were added to the Luria agar)

8. Label the plates:

Label the Luria agar plate with Ampicillin and Arabinose –  Luria+ Amp+Arabinose, your group name, and pGLO E. coli

Label 1 Luria agar plate with Ampicillin –  Luria + Amp, your group name, and pGLO E. coli

Label 1 Luria agar plate with Ampicillin – Luria+ Amp, your group name, and Control E. col

Label the Luria agar plate – Luria, your group name, and Control E. coli

9.  After the 30 minute incubation period, use a sterile transfer pipette to transfer half of the P microcentrifuge tube contents onto the Luria+Amp+ Arabinose pGLO E. coli plate AND half of the P microcentrifuge tube contents onto the Luria+Amp pGLO E. coli plate.

10. Use a sterile spreader to spread the liquid over the surface of both of the pGLO E. coli agar plates. Dispose of the P microcentrifuge tube and the spreader in the autoclave trash.

11.  Use another sterile transfer pipette to transfer half of the C microcentrifuge tube contents  onto the Luria+Amp Control E. coli plate AND half of the C micro centrifuge tube contents onto the Luria Control E. coli plate.

12. Use a sterile spreader to spread the liquid over the surface of both of the control E. coli agar plates. Dispose of the C microcentrifuge tube and spreader in the autoclave trash.

13.  Incubate the inverted plates in the class plate tray until the next lab session.

AFTER INCUBATION-Record the results in the worksheet.

POST TEST

DISCOVERIES IN MICROBIOLOGY

DR. ÉLIE METCHNIKOFF

In 1882, Russian zoologist and microbiologist Dr. Metchinkoff was the first to discover phagocytosis and phagocytes. He advocated the use of lactic acid bacteria (Lactobacillus) for healthy and long life. This became the concept of probiotics in medicine.