Bacterial transformation is a technique widely practiced by scientists for research purposes. This experiment explored the transformation of E. coli cultures with pGLO plasmids to allow the bacterial cells to express a foreign protein and emit a fluorescent glow under UV light. The transformation was completed through the heat shock method. Both transformed and untransformed E. coli cultures were grown in four mediums. The four mediums were made of different combinations of the LB nutrient broth, ampicillin and arabinose C sugar. When exposed to UV light, only the transformed bacteria growing on the LB/ampicillin/arabinose plate had a fluorescent glow.
The pGLO plasmid was genetically engineered to contain the green fluorescent protein (GFP), which was isolated from the jellyfish Aequorea victoria. This jellyfish absorbs blue light from the environment and emits green light in return. Scientist Osamu Shimomura, who first discovered the GFP protein, explained that the peptide chains of these proteins contain a fluorescent chromophore which can be expressed in living organisms1. This revolutionized the field of biomedical research as researchers were able to use this glow-in-the-dark gene to track the activity of proteins inside other living creatures2. Tony Perry and Teru Wakayama at the Advanced Cell Technology in Worcester led a study which genetically engineered mice to express the GFP protein and caused them to glow when hit with UV light3. These mice highlight just how far genetic engineering has come. Prior research indicated that E. coli bacteria can uptake the pGLO plasmid and express the GFP. In this experiment, the E. coli bacteria were transformed using the pGLO plasmid by the heat shock method in order to express the GFP and glow under UV light.
Making the mediums for the bacterial cultures
An agar broth was made by boiling LB agar powder and 500ml of water. A sufficient amount of this solution was poured into two petri dishes labelled “LB -pGLO” and “LB”. Into two dishes labelled “LB/amp -pGLO” and “LB/amp +pGLO”, the agar broth with ampicillin was poured in. Arabinose C sugar was then added to the broth and was poured into one dish labelled “LB/amp/ara +pGLO”(Fig. 1). They were left overnight to harden. Meanwhile, a streak plate was made (Fig. 2).
Making cell membranes more permeable using CaCl2
Two microcentrifuge tubes containing 250?L of the transformation solution (CaCl2) were labelled +pGLO and -pGLO. Using sterile loops, a few E. coli colonies from the streak plate were transferred into the two tubes while making sure there were no floating chunks (Fig. 3).
10?L of the pGLO plasmid were pipetted into the microcentrifuge tube labelled “+pGLO” only. Both tubes were incubated on ice for ten minutes then immediately transferred to a 42°C water bath for 50 seconds and placed on ice for two minutes.
Incubation to allow for bacterial growth
250?L of LB broth were pipetted into the two microcentrifuge tubes and mixed. They were then incubated at room temperature for ten minutes. 100?L from each of the tubes were pipetted into their corresponding plates and was spread on the surface of the agar. The four plates were stacked upside down and left overnight in a 37°C incubator.
All the plates displayed bacterial growth except for the LB/amp -pGLO plate. The different colonies of E. coli could be seen clearly in both +pGLO plates (Fig. 4) whereas in the LB -pGLO plate, there was a lawn growth of bacteria. The mediums of the two +pGLO plates were LB/amp and LB/amp/ara. There were about 200 colonies of E. coli in each of the two +pGLO plates.
When exposed to ultraviolet light, the bacterial colonies in the +pGLO LB/amp/ara plate expressed a fluorescent glow (Fig. 5). Even though there was bacterial growth in two other plates, they did not glow when hit with UV light.
The transformation efficiency, which was the extent to which the E. coli cells were transformed, was calculated and was found to be 1477.7 transformations/?g.
The two pieces of information that were needed to calculate the transformation efficiency were the total number of colonies on the LB/amp/ara +pGLO plate, which was obtained by counting the colonies, and the amount of DNA spread on the agar plate in ?g. To calculate the amount of DNA spread in the plate, the total amount of pGLO DNA in ?g was multiplied by the fraction of DNA used.