April 29, 2024

Artiom Peshkur

10th Grade

Northfield Mount Hermon

Abstract

This research focuses on the extraction of green fluorescent protein (GFP) from Escherichia coli (E. coli) utilizing hydrophobic interaction chromatography (HIC) and recombinant DNA. Despite initial challenges with heat-shocking for the E. Coli to uptake the plasmid, successful culturing in LB broth led to GFP overexpression, as visible under the ultraviolet light. Subsequent centrifugation and cell lysis led to GFP extraction from the supernatant. HIC purification yielded fractions containing GFP, with varying concentrations across samples. This work underscores the viability of GFP as a biomarker for gene expression in biological systems.

Introduction

Green fluorescent protein (GFP) is a protein originally isolated from the jellyfish, Aequorea victoria (1). GFP’s ability to be brightly luminescent under ultraviolet and serves as an excellent reporter of gene expression due to its self-contained chromophore, meaning that there is no need for additional external cofactors of enzymes (2). A plasmid is a circular molecule containing DNA that could be transferred and taken up by host bacteria. (3) A plasmid can be manipulated and a gene can be implanted into it using recombinant DNA technology and procedure, which utilizes restriction enzymes to splice DNA (4). The overarching goal of this experiment is to transfer a pGLO plasmid into a host bacteria such as Escherichia coli (E. Coli) that can overexpress GFP and then extract it. The pGLO plasmid used in the experiment contains the gene for producing GFP, regulating GFP, and for resistance to the antibiotic ampicillin. The E. Coli has to undergo a heat shock for the pGLO plasmids to successfully be taken up. The E. Coli that didn’t take up the plasmid will be killed by the ampicillin and the surviving E. Coli with the pGLO plasmid will produce GFP in the presence of Arabinose. The E. Coli with the pGLO plasmid is then bulk fermented in an LB nutrient broth allowing for the E. Coli to produce vast amounts of GFP. Then lysate was added to break the cell walls of the E. Coli to release the GFP and later isolated through the use of hydrophobic interaction chromatography. To have the highest purity of GFP, column chromatography was used and the fractions were separated and the extracted GFP was visible under ultraviolet light. Monitoring gene expression with a biomarker like GFP is essential for scientists to understand the interactions between proteins in cells.

Methods

I. Inserting the Plasmid

The E. Coli was first streaked across a starting plate to extract a single colony for testing and was incubated for 23 hours at 37°C. Afterward, a single colony was scooped up with a sterile loop and was dispersed in a tube labeled + pGLO that contained 250 μl of transformation solution of 50 mM CaC12. The same process was repeated for a tube labeled -pGLO. Then 10 μl of plasmid was pipetted into the +pGLO tube and then was heat-shocked. Four LB agar plates were prepared with different nutrient components: an LB agar plate (L), 2 LB plates containing ampicillin (LA), and an LB plate containing ampicillin and arabisome (LAA). The E. Coli without the pGLO plasmids were pipetted into agar plates labeled L and LA. The E. Coli with +pGLO plasmids were pipetted into agar plates labeled LA and LAA. The suspension of the E. Coli was evenly spread across the agar surfaces using sterile loops, and the plates were stacked, labeled, and placed upside down in a 37°C incubator for 23 hours. After the overnight incubation, the plates were examined under UV light, and results were documented; however, no +pGLO colonies were visible so a successfully heat-shocked colony was taken from another group (Joey and Annie’s). Then the strand of E. Coli that successfully uptook the pGLO plasmid was cultured with an LB nutrient broth and stored in the freezer at -20°C for 18 days. Once the supernatant containing GFP was removed from the refrigerator and thoroughly thawed, it was centrifuged at 4,500 rpm for ten minutes. A chromatography column was prepared by draining the existing buffer and adding 3 mL of equilibration buffer. Following equilibration, 250 μl of the supernatant was mixed with 250 μl of binding buffer in a new tube. Then the first Eppendorf tube was placed underneath the column and 250 μl of lysate was pipetted into the column. Then 250 μl of the wash buffer was added followed by the TE buffer of 750 μl. Observations were made using UV light to keep track of the GFP and the fractions from the column were collected accordingly.

Results

I. Inserting the Plasmid

II. Cultivating the E. Coli and Extracting the GFP

Discussion

The first key result was the heat shock of the +pGLO plasmids with E. Coli. If the heat-shock was successful there would have been colonies however we can see that there is nothing except air bubbles (Fig. 1 and 2). Therefore none of the strains of E. Coli survived in either the ampicillin or the ampicillin/arabinose LB media agar plate. So a colony that successfully received a +pGLO plasmid was taken from Annie and Joey’s group (Fig. 4). Expected E. Coli growth is visible and the ampicillin was doing its job (Fig. 3). The successfully transferred E. Coli from Annie and Joey’s group was put into an LB nutrient broth, creating an environment. 

The next key result was providing the E. Coli that up took the +pGLO plasmid an LB nutrient broth for the E. Coli to overexpress the GFP (Fig. 5. Afterwards, the small Eppendorf tube was centrifuged at 4500 rpm in a laboratory centrifuge (Fig. 9). Then lysate was added to the Eppendorf tube (Fig. 11) and soon the cells broke and spewed out their contents of GFP under the ultraviolet light (Fig. 10). 

The last key result was successfully filtering GFP from the supernatant utilizing hydrophobic interaction chromatography (HIC). The HIC successfully worked to purify the GFP from the supernatant (Fig. 6). Results were gathered and collected into fractions (Fig. 7) and fraction (tube) #4 had the highest concentration of GFP because the TE buffer allowed the GFP to pass through the matrix of the column at once. Fraction #4 had some, though a significantly lower, concentration of GFP because some of the GFP flowed out of the column as the 250 µl of wash buffer was added. 

The first error was that the heat shock was at first unsuccessful. This was probably because the heat change wasn’t rapid enough to cause the E. Coli to take up the +pGLO plasmid, and since the tubes weren’t deep enough in the ice. However, upon reexamination of the agar LAA agar plate after six weeks, there were E. Coli colonies that up took the +pGLO plasmids along with another colony on the side of the plate that came from contamination (Fig. 12). So it wasn’t that the heat-shock was unsuccessful, it was the fact that only a small amount of E. Coli cells absorbed the encoded plasmid. To further explain Figure 12, the older cells on the innermost part of the colony had their GFP degraded, while the newer cells had their GFP intact and fluorescent. Fortunately enough, by taking a colony from someone else, the experiment proceeded as planned, as demonstrated by the key results. Further research should be done to reach accurate conclusions.