Bio Nerds: pGLO Transformation Lab

Hello and welcome to our fabulous P.Glo lab done by Aliya, Emma, Cara, and Ally (your fav senior ladies who value and appreciate you Mr.Filipek)

Purpose

The purpose of this experiment was to perform and prove genetic transformation which is insertion of a gene into an organism in order to change it's trait. In this experiment we were testing the insertion of the GFP(green fluorescent protein) gene into the P.Glo plasmid of the E.coli cells.

Introduction

In this experiment, E.coli is a bacteria that is commonly found inside of mammals and birds and is used to breakdown food into small molecules that can be transferred into your blood. It is a very basic prokaryotic cell and only lives a short time. Most types of E.coli are good for you but there are a very few select types that can be poisonous to humans such as E.coli found in raw meat. Genetic transformation, in this experiment is used to transfer new DNA from the GFP to plasmid of the E.coli cells. A plasmid is a small circular piece of DNA where the GFP can be inserted in order to show new traits within the cell. The insertion of GFP into the pGlo makes the cell resistant to antibiotics.

Methods

We started of by using two micro test tubes one of +pGlo (containing pGlo) and one of -pGlo (not containing pGlo). We then placed 250 microliters of CaCl2 into each tube. Next the two tubes we're placed on ice. After this, we put a sample of the E.coli bacteria into each tube using a loop. Immediately, we inserted pGlo plasmid DNA into the positive pGlo tube but not in the negative pGlo tube. Then we incubated (let them sit on ice) for 10 minutes. Right after this, we performed a heat shock on both tubes. Next, we put 250 microliters of LB nutrient broth in both tubes. We then placed 100 microliters of the solution from each tubes onto 2 Agar plates each, resultin in 4 total plates. One plate of +PGlo LB/amp, one of +pGlo LB/amp/ara, one of -pGlo LB/amp, and one of -pGlo LB.

Data

Graphs and charts

Drawn-out representations of our Agar plates and their contents after the lab was completed

Discussion

Our results from this lab were a direct result of the proccess of genetic transformation. Some of the dishes expressed the pGLO trait while others did not because of this proccess. Those that did express the trait those that expressed the "glow in the dark" trait were those with pGLO and LB/amp. The plate with LB/amp/ara contained more colonies because of the sugar that was added to the plate that helped with growth. If we were to make adjustments to this lab, we would put more of each substance of each plate in order to see more colonies then what we did the first time. It's difficult to tell whether our results are completely accurate due to how little of data that we have. As predicted, the petri dishes that had the GFP inserted into the plasmid glowed in the dark, while the petri dishes without the plasmid did not. The insertion of the GFP into the plasmid then makes it resistant to antibiotic. The LB plate has no growth because it has no protein to resist antibiotic.

Conclusion

Through this lab we were able to prove that pGLO causes the bacteria to be able to resist the antibiotic.

References

http://scienceforkids.kidipede.com/biology/cells/ecoli.htm