Changing E. Coli Using Plasmid DNA Essay Example

📌Category: Biology, Genetics, Science
📌Words: 1278
📌Pages: 5
📌Published: 11 June 2022

Introduction

This practical is about the changing of e coli using pGLO plasmid DNA. Doing will allow us to observe the physical changes that might occur when doing this to the e coli. The outcome for this experiment is to see if we can add plasmid DNA pGLO to the bacteria E. coli to see if do this we add the plasmid to one sample of e. coli and the other sample we didn’t add the plasmid to see if the experiment worked. The plasmid DNA pGLO contains the ampicillin resistance gene or bla gene which encodes a b-lactamase which is secreted by the bacterium and can destroy penicillin-type antibiotics by the process of hydrolysis. pGLO also contains the GFP gene is cloned from the jelly Aequorea Victoria and it’s a green fluorescent protein. Articles have been written on GFP for more information on the protein For example, ‘GFP became a well-established marker of gene expression and protein targeting in intact cells and organisms. The engineering of GFP into chimeric proteins opens new vistas in physiological indicators, biosensors, and photochemical memories’ (Tsien, 1998) Ori is part of pGLO. It means the origin of replication allows the plasmid to replicate without the bacterial DNA. There is also AraC which is an arabinose promotor protein that drives transcription of the GFP gene, which is related to the promotor driving the arabinose operon. AraC is expressed because of arabinose. The pGLO plasmid glows under UV light so if it’s present, we will be able to see it under UV light. The aim of this practical is to transfer the plasmid DNA into bacterial cells. To do this the DNA must cross the cell membrane into the bacteria cell but to do this another step must be done as this step alone would not be highly effective. 

Method 

First, the closed microfuge tube was labelled with the person who is conducting the experiment initials on the ¬+ pGLO and -pGLO tube and is then placed in the foam rack. Then the tubes were opened, and the sterile pipette is used to transfer 250l of transformation solution also known as CaCl2 into each tube after the tubes were placed on ice for 5 minutes. The Sterile loop is used to pick up a single colony from a plate with E. coli colonies. Following this + pGLO tube is picked up and the loop is immersed in transformation solution at the bottom of the tube. Then the loop spun between index finger and thumb until all the colony was dispersed into the transformation solution. After the tube was placed back into the rack in the ice. The loop that was used is then disposed of into the disposal jar that was provided. Afterward, a new sterile loop was used, and the procedure was repeated was done for the -PGLO tube. The new sterile loop was immersed into the plasmid DNA solution. A loopful of the solution was removed and mixed into the suspension of bacteria in the + pGLO tube. The tube was then closed and was returned to the ice. The plasmid was not added to the -pGLO tube, it was left on ice with the cap closed. The tubes were incubated on ice for 10 minutes it was ensured the bottom of the tubes were in contact with ice. While the tubes were in the ice the four agar plates were labelled with the persons’ initials and the first agar plate was labelled with LB/amp +pGLO the second plate was labelled with LB/amp/ara +pGLO the third plate was labelled with LB/amp -pGLO. Then the final plate was labelled with LB -pGLO. After 10 minutes of incubation on ice both +pGLO and –pGLO were inserted into the foam float and the float with the tubes was placed in a water bath set at 42°C and was placed in the water bath for 50 seconds. The tubes were immersed in the water. After 50 seconds the tubes were placed back onto the ice The tubes were left on the ice for 2 minutes and then the foam rack containing the tubes was moved onto the benchtop. The tubes were then opened, and a new sterile pipette tip was used to add 250uL LB nutrient broth to the tube and was reclosed. This was then repeated with a fresh sterile pipette tip for the other tube. The tubes were then taped to mix the bacteria in the LB broth, and it was incubated in the tubes for 30 minutes in the shaking incubator at 37°C. After 30 minutes the closed tubes were taped to resuspend the bacteria. Then 100 uL of the transformed bacteria were transferred onto each of the two plates labelled +pGLO. After 100 uL of control bacteria was placed onto each of the two plates which were labelled -pGLO. A new sterile loop was used for each plate and the loop was used to spread the suspensions evenly around the surface of the agar plate. The loop passed the surface of the agar plate back and forth. The plates were then staked and were taped together. Afterward, the plates were handed to the demonstrator who then placed them in an incubator at 37 °C, where they were left overnight and were transferred to a cold room so they can be stored until analysis in practical 2.

Results

One of the plates that have the plasmid and Amp (ampicillin resistance gene) and there was growth, but it was white instead of having a green fluorescence. Plate two had Amp/Ara and the plasmid and there was growth and green fluorescence. The third plate has Amp and no plasmid and there was growth but there was no fluorescence. The final plate has no Amp/ara and no plasmid so the results for this are no growth and no green fluorescence.

Selection Amp Amp/ara Amp None

Plasmid + + - -

Demonstration plates

Growth but are white so have no fluorescence Growth and fluorescence Growth but no fluorescence No growth or fluorescence 

Discussion 

The results that were obtained show that having plasmid and arabinose (ara) affect the growth and whether there is fluorescence or not. One plate with plasmid amp/ara shows this as it is the only plate with fluorescence, suggesting that plasmid and arabinose changed the bacteria. This is reinforced by one of the plates where amp and the plasmid were used, and the results showed that there was growth but there was no fluorescence suggesting that there needs to be arabinose and plasmid for there to be a green fluorescence and that the Amp might affect whether there is fluorescence or not.

The molecular mass of the plasmid pGLO is around 3300kD. We used pGLO because ‘pGLO transformation experiments can be used to explore aspects of the system in more detail and to demonstrate other biological concepts’ (Deutch, 2019). The plasmid charge is negative because plasmids have a phosphate group on each nucleotide giving it a negative charge. As the plasmid is a charged molecule it cannot pass freely through the cell membrane of the bacteria as it would have to pass through a protein channel by the process of facilitated diffusion which means that large, charged, ion-molecule that cannot pass through the cell membrane so it must pass through a membrane protein. The molecule moves down its concentration gradient, so it does not require ATP (energy). The purpose of ampicillin is to create a medium that allows only cells containing the plasmid pGLO to grow on the agar plate. The purpose of the arabinose medium is to induce the expression of GFP by binding to the protein AraC. This then can allow the growth of both pGLO and non-pGLO cells to grow but only the pGLO cells will become fluorescent. The plate with Amp/ara and has the plasmid has green fluorescence and had the most growth. The fact that there is green fluorescence for this plate suggests that GFP is being expressed.

In conclusion, what is learned from this practical is that varied factors affect whether the E. coli bacteria can be changed.

References

Deutch, C. E. (2019). Transformation of Escherichia coli with the pGLO Plasmid: Going beyond the Kit . The American Biology Teacher , 52–55.

Tsien, R. Y. (1998). THE GREEN FLUORESCENT PROTEIN. Annual Review of Biochemistry, Vol. 67:509-544.

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