Jason

Daniel

Zach

Jordan

Purpose: The purpose of this lab was to study genetic transformation by making antibiotic resistant and glow in the dark E. Coli.

Introduction:

Genes are pieces of DNA within an organism that provide the codes for specific traits. A change caused by genes is called genetic transformation, which involves the insertion of a gene into an organism which changes the organism’s trait. In the E Coli experiment, colonies of the E Coli bacteria will be transformed with a gene that codes for Green Fluorescent Protein (GFP), the same gene that causes bioluminescent jellyfish to glow in the dark. This gene will cause the transformed bacteria to glow underneath an ultraviolet light. Due to the fact that plasmid DNA usually contains genes which code for traits that will be beneficial to an organism’s survival, the plasmid which is being used also contains pGLO, which allows the bacteria to resist the antibiotic ampicillin.

Methods:

First, we labeled two test tubes; one reading +Pglo to represent the microtube that will include the Pglo plasmid DNA. The other reading -Pglo to signify the absence of the Pglo plasmid DNA.

+Pglo Microtube

Next, we used a sterile pipet to administer 20 μL of transformation solution into both tubes. We promptly put the microtubes on ice.

-Pglo Microtube

+Pglo and -Pglo microtubes filled with transformation solution on ice.

We then used a sterile loop to scrape a colony of E Coli from the starter plates and add to the transformation solution in both microtubes. We spun the sterile loop in the solutions of both microtubes to ensure the bacteria had been thoroughly incorporated into the solutions. Next, we administered the UV light to the microtubes to ensure that E Coli does not glow naturally. We then put the tubes back on ice.

Shining UV Light onto Micro Tubes after E Coli colonies had been distributed.

Once we made sure E Coli does not glow naturally, we used a separate sterile loop and used it to include Pglo Plasmid DNA into the microtubule labeled +Pglo. Shortly thereafter, we used the UV Lamp once again on the microtubules and observed that the solution in the +Pglo Microtube glowed, while the -Pglo microtube did not. We then placed the microtubes back on ice for 10 minutes.

The solution of the +Pglo Microtube glows while the -Pglo microtube does not.

Microtubes placed back on ice for 10 minutes.

We then administered the UV Light to the Pglo plasmid DNA to observe that it does glow on it’s own naturally.

Plasmid DNA does glow naturally.

While the microtubes sat on ice, we grabbed four plates of LB nutrient agar, a medium used to culture more bacteria, in this case E Coli. The plates were labeled -Pglo LB, -Pglo LB/Amp, +Pglo LB Amp, and +Pglo LB/Amp/Ara.

Ara - Arabinose

Amp - Ampicillin

Labeled LB Nutrient Agar Plates

After the 10 minutes had passed, we removed the microtubes from the ice and put them in 42 Degree Celsius water. The Microtubes floated in the hot water for 50 seconds.

Microtubes introduced to water at 42 degrees celsius.

After 50 seconds in the water, the tubes were returned to the ice cup. The hot water made the E Coli membranes permeable to the Plasmid DNA and once the Microtubes were put back on ice the membranes began to seal. Then, we added 250 μL of nutrient broth into each tube, stirred the solutions and let them sit for 10 minutes.

After 10 minutes, we used a sterile pipet to administer 100 μL of the solutions onto their respective plates.

+Pglo - (LB/Amp),  (LB/Amp/Ara)

-Pglo - (LB),  (LB/Amp)

100μL of each solution being pipetted onto their own plates.

Using a sterile loop to efficiently spread the bacteria into the plate solution.

The Plates were then sealed off and placed upside down due to perspiration building on the top of the lid otherwise. They were then left alone for a day in order for the bacteria to grow in the medium.

Plates Incubating

24 hours later, the plates were unbundled, uncapped, and observed. We found that of the -Pglo tubes, the plate with only LB underwent significant growth with bacteria spread across the plate and the plate with the LB and Ampicillin showed -growth due to the antibiotic killing off the E Coli. Of the +Pglo tubes, the plate with the LB and Ampicillin showed minor growth, signifying that the E Coli had become antibiotic resistant. The Plate with the LB, Ampicillin, and Arabinose glowed and showed minor growth due to the acquired antibiotic resistance and a successfully implemented gene causing the bacteria to glow in the presence of Arabinose.

+Pglo - (LB/Amp/Ara) glowing due to the implemented gene.

After we gathered our data, all of the bacteria was annihilated by bleach baths and all materials were safely disposed of.

Data:

Discussion:To successfully move the pGLO plasmid DNA through the cell membrane of the E. Coli we had to do several steps. First we used the transformation solution calcium chloride to assist in bacterial transformation by puncturing the cell membrane of the E. coli. Then we placed the E. coli in hot water treating it with heat shock. The heat shock changes the liquidity of the cell’s membrane making it easier for DNA to get into the cell.  

Conclusion:

We experimentally proved that transformation is an important aspect of genetics. Incorporating segments of DNA into other organisms has the potential to be extremely useful. In this particular case, the segment of the DNA coded for the ability to glow in the dark when exposed to ultraviolet light and antibiotic resistance. The results of the experiment show that the GFP is only responsible for making the jellyfish glow when in the presence of the sugar arabinose. When sugar arabinose wasn't present E. coli did not glow in the dark.