Pglo Lab Fun!!

Overview

For this lab, our group transformed bacteria using a plasmid, with the objective to make the bacteria glow in the dark. The plasmid contained the gene for GFP (green fluorescence protein), for beta-lactamase, and also contained an inducer that was turned on by arabinose, and triggered production of GFP. In order to quantify our results, we then determined the transformation efficiency of the procedure (how effective our transformation actually was relative to the number of bacteria).

In order to gather our results and data, we observed data such as the amount of growth on each plate, the color of the bacteria, and whether the bacteria glowed in the dark or not.

We expected that the only plate that would contain glowing bacteria was the +lb/amp/ara plate, because it contained lb (to allow the bacteria to grow), and the pGLO plasmid (which helped neutralize ampicillin), and arabinose, which served as an inducer to turn on the GFP gene.

pGlo gene

Materials

E. coli starter plate (1)
Poured agar plates (1 LB, 2 LB/amp, 1 LB/amp/ara) (4)
Transformation solution (1)
LB nutrient broth (1)
Inoculation loops (7) (1 pk of 10)
Pipets (5)
Foam microtube holder/float (1)
Container full of crushed ice (foam cup) (1)
Marking pen (1)

Rehydrated pGLO plasmid (1 vial)
42°C water bath and thermometer (1)
37°C incubator



Procedure

1. First, we used the marking pen to label each tube +pGLO and -pGLO.

2. We then used a new pipet to add 250 microliters of transformation solution to each of the tubes (calcium chloride)

3. After placing the tubes on ice, we used a sterile loop to carefully pick up a single colony of bacteria from our starter plate. We then carefully placed it into the -pGLO tube, spinning the loop until the entire colony was in. We then repeated for the +pGLO tube.

Adding the e. coli

4. We then went over to our instructor Mr. Wong, where he added the plasmid to the +pGLO tube.

5. We then incubated our tubes on ice for 10 minutes. While we were waiting, we examined our plates that were labeled -lb, -lb/amp, +lb/amp/ara, +lb/amp.

6. Using the foam rack as a holder, we put the tubes into a hot water bath (42 degrees Celsius) for exactly 50 seconds. After the heat shock, we immediately transferred the tubes onto ice.

7. After removing the tubes from the ice, we placed them back on the test tube rack at room temperature. We then added 250 microliters of LB nutrient broth to each test tube. Then, we incubated the tubes at room temperature for 10 minutes.

8. We tapped the tubes to mix them. Using new pipettes, we piped 100 microliters from each test tube into their respective plates. We added the contents of the +pGLO tubes to the plates labeled +, and the contents of the -pGLO tubes to the plates labeled -.

9. We then used a new sterile loop to "spread" the bacteria around the plate. We spread them in four quadrants in a zigzag pattern.

Spreading the bacteria

10. We then stacked our plates together and taped them together, and gave them to Mr. Wong, who incubated them for a day.

Data/Explanation of Data

Table 1 

	-lb	-lb/amp	+lb/amp	+lb/amp/ara
Color	white	white	white	White
Rate of growth	Lawn of growth	No growth	growth	growth
Glow	no	no	no	yes

As can be seen from Table 1, the only plate where glow was observed was the plate with +lb/amp/ara. The color of the bacteria did not significantly change over the course of transformation. The only place where there was no growth was the -lb/amp plate. We concluded that this occurred because it didn't have the pGLO gene that contained the beta-lactamase gene that would destroy the ampicillin; which was allowed to destroy the bacteria unhindered, and therefore was not resistant to antibiotics. The bacteria in the + plates were antibiotic resistant, and therefore had growth. The LB broth served as a growth medium in this experiment. In the -lb plate, because there was no ampicillin to kill the bacteria, there was a lawn of growth. In the -lb/amp plate, there was no growth even though there was LB because the ampicillin killed it. In the +lb/amp plate, there was growth because LB was present, and the plasmid helped the bacteria neutralize ampicillin, which also occurred in the +lb/amp/ara plate. As we expected, the only plate that contained glow in the dark bacteria was the +lb/amp/ara plate. This occurred because the bacteria was allowed to grow (because of the lb), and the plasmid allowed it to destroy the ampicillin, and the arabinose activated the GFP gene [as an inducer], which produced the protein to make the bacteria glow in the dark. 

Analysis and Takeaways

What I definitely enjoyed most about this experiment was working with the bacteria. I have had almost no experience actually handling bacteria, and this lab allowed me to tackle this experience head on. I particularly enjoyed spreading the e. coli on the plate. My most valuable takeaway from this experiment was a new understanding of transformation and the concept of a gene. We successfully inserted the plasmid, a secondary form of DNA for bacteria. The bacteria glowed in the dark because the pGLO gene produced GFP, which caused the observed change in phenotype. From this lab, I refined my concept of how a gene translate into a protein. In addition, another valuable takeaway from this lab was real life application, because biotechnology nowadays concentrates heavily on the concept of the gene.
 I can begin to see how much easier transformation would be for a large lab. For example, a large lab could possibly easily transform a bacteria to produce insulin, and then bottle and sell the insulin. Reflecting on this experiment, we could have easily increased our transformation efficiency by more carefully completely immersing the plasmid in. In addition, we could have possibly transferred the tubes from the heat bath to ice more quickly. We could have altered this experiment by possibly using other ways to crack open and reseal the plasmid. 

My questions left after this experiment are

Are genetic transformation of animals and human ethical?

How did they calculate the needed times for the heat shock/cold shock? (e.g. 50 seconds and 10 minutes)