The bacterial transfer of genetic material requires either a small fragment of DNA or a plasmid. Plasmids are small DNA molecules that replicate on their own in the bacterial cell. They are used in recombinant DNA technology for many reasons. One reason why they are used is to produce large amounts of needed proteins such as human growth hormone.

In the pGLO plasmid, there are 2 genes that can be incorporated into a transformed bacterium (bacteria that have plasmids in them), bla and GFP. bla is the gene that that allows for betalactamase activity, an activity that allows the bacteria to have ampicillin (an antibiotic) resistance. This gene is continuously expressed in the bacteria (E. coli). GFP (green fluorescent protein) is a gene that is regulated by a repressor (a protein that prevents the expression of a gene) called araC. GFP can only be activated when the inducer, a sugar called arabinose, is present. To make the bacteria glow, the sugar, arabinose will be used to activate the expression of GFP.

The bacterial cells used in this transformation must be made competent or they must be chemically or electrically activated to create conditions where they will incorporate external DNA into their cytoplasm before they can be used. One can order competent bacterial cells from a biotechnology lab.

This is a list of the materials needed in order to conduct this experiment:

1. Competent E. coli

2. pGLO plasmid

3. Transformation Solution (CaCl2)

4. LB broth (Lysogeny broth, a nutritionally rich medium primarily used for the growth of bacteria)

5. water bath set at 42ᵒ C

6. Ice

7. Fixed pipettor tips

8. 2 centrifuge tubes labeled: +pGLO and -pGLO

9. Centrifuge tube holder

10. 2-LB/amp plates

11. 1-LB plate

12. 1-LB/amp/ara plate

13. Inoculating loop

14. UV light

Methods

1. Pipette 250μl CaCl2 into each centrifuge tube

2. Transfer a colony of E. coli into each tube and disperse cells by tapping gently on the tube then place it in ice.

3. Using an inoculating loop Add 1 loopful of pGLO plasmid to the +pGLO tube and then put both tubes on ice.

4. Place the tubes in the tube rack and heat shock for 50 seconds in the water bath. Then immediately return them to the ice for 2 minutes.

5. Add 250μl LB broth to each tube and incubate them at room temperature for 10 minutes. While waiting label your plates as follows:

·         +pGLO LB/amp

·         +pGLO LB/amp/ara

·         -pGLO LB/amp

·         -pGLO LB

6. From the +pGLO tube, pipette 250μl of solution onto the +pGLO, LB/amp plate, and 250μl of solution onto the +pGLO, LB/amp/ara plate. Then use a sterile loop to spread the bacterial solution around the plate.

7. From the -pGLO tube, pipette 250μl of solution onto the -pGLO, LB/amp plate, and 250μl of solution onto the -pGLO, LB plate. Then use a sterile loop to spread the bacterial solution around the plate.

8. Incubate at 37ᵒC overnight.

9. The next morning check the plates for growth and whether or not they glow under UV light

10. You should see:

·         +pGLO LB/amp – growth (is transformed and has resistance to ampicillin)

·         +pGLO LB/amp/ara – growth and glowing because sugar is present (Is transformed and the sugar is present to make it glow)

·         -pGLO LB/amp – no growth (The antibiotic is present and it has no ampicillin resistance)

·         -pGLO LB – growth (no antibiotic or ampicillin resistance is present)

In the cells that glow, the pGLO plasmid which was once found in jellyfish is now incorporated into the bacterial cells.