Essay: Transforming E. coli with pGLO, Express GFP and Purify with HIC: A Guide



Transformation and Lysis of pGLO to Purify and Express Green Fluorescent Protein

Abstract

The purpose of this experiment was to transform E. coli bacteria with pGLO plasmid, express the Green Fluorescent Protein (GFP) and purify the extracted GFP. This was done by using techniques such as transformation, transformation efficiency, lysis, and hydrophobic interaction chromatography. The results show that GFP was successfully purified from the transformed E. coli bacteria with the help of HIC.

Introduction

One of sciences most important innovations is the ability to introduce DNA into a bacterial cell. This has great practical importance in many different ways. This transformation has great importance in the genetics and molecular biology. Few species of bacteria such as Haemophilus influenzae (VanWagoner et al. 2004) and Streptococcus pneumoniae (Battig and Mulemann 2008), have been found to have this ability to take up foreign DNA. A large majority of bacterial species are not able to take up DNA naturally. Recently, scientists discovered the Yoshida effect (Yoshida 2007) which describes the process of altering bacteria’s genetic properties by using a number of techniques such as using sliding friction force and nanosized articular materials (Plasmid uptake by bacteria).

Green Fluorescent Protein can be derived from few species. More frequently it is derived from the jellyfish Aequora victoria and the sea pansy Renilla reniformis (Green Fluorescent Protein). This protein is studied for its transformative properties and its ability to be expressed in many different types of cells. GFP was able to be expressed in different yeast, mammalian, and bacterial cells (Green Fluorescent Protein). These initial experiments showed that GFP could be used as a noninvasive way to monitor gene expression within these different types of cells. The new-found ability to transform a cell with foreign DNA containing GFP opens a new idea that other plasmids with other proteins and genes can be used to transform a cell. These findings could allow new cancer research that involves the transformation of cancerous cells with DNA that has a cancer fighting gene or protein. Therefore, killing the cancerous cells.

Hydrophobic interaction chromatography is a separation technique that uses the properties of hydrophobic compounds to separate proteins from one another (Hydrophobic Interaction Chromatography). In this experiment, GFP was separated from other compounds because of its hydrophobic properties. GFP’s hydrophobic amino acid side chains interacted with the hydrophobic groups on the column apparatus used. This allows it to be separated from the other compounds with different properties.

Materials and Methods

Two microfuge tubes were labeled “pGLO+” and “pGLO-” and 250 L (transformation solution) CaCl2 was added to each. The tubes were placed on ice. Four large E. coli colonies were taken on a sterile loop from the upper area of the starter plate and swirled into each microfuge tube. 10 L of the pGLO plasmid was pipetted into the “pGLO+” tube and both tubes were incubated on ice. Four plates were labeled the following: “LB/amp: pGLO+” “LB/amp/ara: pGLO+” “LB/amp: pGLO-” “LB: pGLO-”. After the incubation, each tube was heat shocked at 42C for exactly 50 seconds and then immediately returned to the ice for 2 minutes. LB broth (250L) was added to each tube and they were incubated at room temperature for 10 minutes. The bacterial suspension (100L) was added to each correlating plate and incubated at 37C overnight. After returning the following day, the plates were examined, and no colonies were found. After an extra day of incubation, the plates were moved to the fridge.

The next week, the transformation efficiency was found from the colonies at the next table. The transformed E. coli plate (LB/amp/ara: pGLO+) was observed under UV light to identify a single growing colony. The same was done for the non-glowing LB/amp plate. Two 15mL culture tubes with 2mL growth media were labeled “+” and “-”. The glowing colony was added to the + tube and the non-glowing colony was added to the – tube via sterile loop. Both tubes were shaken vigorously by hand and put in the shaking incubator overnight. The next day, the cultures were transferred to the fridge.

The next week, the cultures were removed from the fridge and observed under UV light. The “-” tube was discarded and 1.4 mL of the “+” tube was added to a microfuge tube. The culture was spun in the balanced centrifuge for 5 minutes. The supernatant was poured back into the original + tube and the bacterial pellet was observed under UV light. Tris-EDTA (TE) buffer was added to the pellet and resuspended via vortexing. Then, 20uL of lysozyme was added to the solution.  The newly resuspended solution was observed under UV light and incubated at room temperature for 15 minutes. The tube was placed in the freezer, to enhance lysing, for a week.

The samples were thawed at room temperature for several minutes and then vortexed and observed under UV light. The samples were spun in a balanced centrifuge for 10 minutes. During the centrifuge time, the chromatography column was prepped. The separated supernatant and pellet were observed under UV light. Then, 250 L supernatant and Binding Buffer (BB) was transferred into a new tube labeled + and lightly mixed via pipette. Three collection tubes were labeled 1,2,3. All 500 L of solution were drained into tube 1. Next, 250 L Washing Buffer (WB) was drained into tube 2. Lastly, 750 L Elution Buffer (EB) was drained through the column into tube 3. All three tubes were examined and observed under UV light.

Results

The purpose of this experiment was to transform E. coli bacteria with plasmid that contained GFP and then purify the expressed GFP using Hydrophobic Interaction Chromatography. After growing the E. coli, the growth and color of colony were predicted. All plates were expected to grow except for the -pGLO LB/AMP plate. This is because the ampicillin antibiotic was inhibiting the growth of the bacteria (Figure 1). All of the plates were expected to be clear under UV light except for the +pGLO LB/AMP/ara plate. This is because the arabinose nutrient helped turn on the GFP while also allowing the bacteria to grow (Figure 1).

In week two, Growth and Determination of Transformation Efficiency, the results all show the expected results discussed prior. The +pGLO LB/AMP plate contained 71 colonies of E. coli. The +pGLO LB/AMP/ara plate contained 53 colonies of bacteria. The -pGLO LB/AMP plate had no growth and the -pGLO LB plate had an entire lawn of bacterial growth. These numbers were then used to calculate a 3.38×102 transformation efficiency with an error of 42.25% (Table 2).

During week 3, the sample was manipulated several times. After centrifuging the mixture, the first time, the supernatant was clear, and the pellet was green and glowing under the UV light (Table 3). After adding the TE buffer and mixing, the pellet was broken up and floating in the supernatant. The particles were glowing throughout the supernatant, but the supernatant remained clear (Table 3). After resuspending the mixture and lysing the bacteria, the GFP was extracted from the bacteria and made the whole mixture glow (Table 3).

Hydrophobic interaction chromatography was used to separate the GFP from other proteins and molecules in the solution. After the first wash with the binding buffer and supernatant, the solution was clear under UV light (Figure 1). After the second wash with the washing buffer and hydrophobic remanence, the solution was clear under the UV light (Figure 1). After the third wash with the elution buffer, the solution was a green color and glowed under the UV light (Figure 1). This shows that the GFP was isolated and expressed.

Plate Plasmid Condition Growth/No Growth Color of Colony

+pGLO LB/AMP Present Growth Clear

+pGLO LB/AMP/ara Present Growth Green

-pGLO LB/AMP Not present No growth Clear

-pGLO LB Not present Growth Clear

Table 1: Results of Transformation of pGLO plasmid

Plate Condition Growth? Color # of Colonies

+pGLO LB/AMP Present Growth Clear 71

+pGLO LB/AMP/ara Present Growth Green 53

-pGLO LB/AMP Not Present No growth Clear 0

-pGLO LB Not Present Growth Clear Lawn

Transformation Efficiency 3.38×102 Error   42.25%

Table 2: Results of Growth and Determination of Transformation Efficiency

Condition

# Pellet or Solution Color/Result

1 Supernatant after first spin Supernatant = clear

Pellet = green, glowing

2 Bacterial pellet after adding TE buffer Pellet = glowing particles throughout mixture

3 After lysozyme and resuspension Solution/Mixture = green, glowing

Table 3: Results of Bacterial Concentration and Lysis

Figure 1: Results of Purification of GFP using HIC

Discussion

The purpose of this experiment was to transform E. coli bacteria with pGLO plasmid, express the Green Fluorescent Protein (GFP) and purify the extracted GFP. The results show that while the E. coli were not very competent in transforming with the extracellular DNA (Table 2), some of the bacteria was able to be transformed. Also, the green fluorescent protein from the plasmid, was successfully purified (Figure 1).  These results are similar to the Green Fluorescent Protein experiment and the Hydrophobic Interaction Chromatography experiment because GFP was successfully taken up by bacterial cells and then purified using the HIC technique. In comparison to the Plasmid uptake by bacteria experiment by Yoshida, the transformation experiment conducted in the King’s College lab was less successful than the experiment Yoshida conducted using the Yoshida method. This is known because the transformation efficiency was better in the Yoshida experiment.

A possible source of the 43.25% error in transformation efficiency could have been from the methodology of the lab or human error. A lack of transformation solution could have restricted the number of bacterial cells that were transformed. Multiple solutions and bacterial plates were used in this lab. It is possible that cross contamination occurred. Disposable pipettes and sterile loops were used to reduce the risk of cross contamination but, there is always a chance that they could’ve been cross contaminated before used in the lab, or that a new substance, such as a different species of bacteria, was introduced. This would be very hard to prevent because sterile techniques are already being used in the laboratory. Transformation efficiency was used to measure how successfully the plasmid was introduced into the bacterial cells. This calculation depends highly on the number of bacteria taken from the starter colony that was grown in the first week of the lab. Only one colony was taken from the sample of 53 colonies grown. It is very unlikely that all colonies on the plate were identical in number of cells or the efficiency of the specific cells to transform.

This lab implies that genes can be taken from one source and carried anywhere.  While there are exceptions to this, the general idea could be used to help many people. This general idea could be tweaked to certain genes or cells to help cure genetic diseases such as different types of cancers, Parkinson’s disease, Tay-Sachs disease, Crohn’s disease, and cystic fibrosis. While there are limited cells that have been found to have transformative properties, there are constant advances in the medical world that could one day allow for a cure to these diseases.