Bacterial Transformation Lab

Last Wednesday (January 16, 2013) I couldn't go to my internship because of the heavy snow:(

However, luckily, we were doing a bacterial transformation lab in my AP biology class, and it is basically what my internship is doing. Transformation is when bacteria intake a piece of foreign gene and express the new gene. In class, we inserted an engineered plasmid (pGLO)containing both a gene that codes for Green Fluorescent Protein (GFP) from a bioluminescent jellyfish and the gene for antibiotics (ampicillin) resistance into E. coli. The transformed bacteria could grow on LB plate (minimum nutrient) with ampicillin and would glow under UV light if arabinose (a type of sugar) is provided.

The brief procedures are:

1. Label the two micro test tubes "+pGLO" and "-pGLO".
2. Use pipettes to transfer 250μl of transformation solution (CaCl2) into both test tubes. 
3. Use a sterile inoculation loops to pick up a single colony of E. coli and added to the test tubes (one colony in each by using a new loops every time). Then, place the tubes in the ice (It was funny how we just walk out and fill the cup with a pile of snow!).
4. Use a new loop to withdraw a film of plasmid and add it to the "+pGLO" tube but not to the other. Softly mix the solution.
5. Place the tubes in ice for 10 minutes.
6. Meanwhile, label the four agar (nutrient) plates: "+pGLO LB/amp/ara", "+pGLO LB/amp", "-pGLO LB/amp", and "-pGLO LB"
7. After 10 minutes, quickly transfer the tubes into the 42C water bath, a process called "heat shock". Leave the tubes in water bath for 50 seconds, and place them back on ice for 2 min.
8. Use a new sterile pipette for each tube and transfer 100μl to the appropriate plate. 
9. Use a new loop for each plate to spread the suspensions evenly on the surface of the agar. Close the plates and check the result after a few hours.

At (5) and (7) when we put the bacteria in ice, they close their pores and go to dormant mode. When we heat shock them, their pores suddenly open and take in the inserted DNA. Thus, it is very critical to follow the protocol during heat shock steps.

The next day we checked our results!!

There are "colonies" of E.coli on both +pGLO LB/amp/ara and +pGLO LB/amp plates. We know those bacteria are transformed because they are antibiotic resistant. Yet, only bacteria on +pGLO LB/amp/ara can glow under UV light because they were given arabinose. There is bacteria "lawn" in -pGLO LB since the plate contains no ampicillin and any bacteria can live. Plate -pGLO LB/amp was clear because none of the bacteria were transformed, and thus, they are not antibiotic resistant.

This lab demonstrates one of the major techniques we use in biotechnology. It also shows how amazing the universality of genetic codes are!

Lastly, this is my glowing bacteria!!:D

This Wednesday we are having a school service day, so I won't be able to go to my internship. However, I am very excited about what I am going to do next!:)