Abstract
While the bacterium Escherichia coli has pathogenic strains that cause food poisoning and illness, they are mostly harmless and play an important role in digestion. Due to E. Coli's strains having such a diverse nature, they can be manipulated and are often used as hosts for testing foreign DNA sequences in experiments such as the one presented. The aim of the experiment presented is to evaluate the bacterium’s ability to intake two forms of plasmids, plasmid lux and pUC18, which carry ampicillin resistant genes, and observe its capability to survive in an ampicillin rich environment once inserted with the ampicillin-resistance gene.
Prior to the introduction of the plasmids to the bacterium, steps were taken to ensure that the plasmids could be successfully introduced into the E. coli. First, Calcium Chloride was added to the E. coli to increase it’s competency, or overall ability to intake the plasmid DNA. The Heat Shock method was also applied to facilitate the introduction of the plasmids into the competent cells. After the transformation of the bacterium, it was placed in an ampicillin rich environment and it’s growth patterns were observed. Agar served as a growth medium in which the transformation efficiency could be tested and observed. The Lysogeny Broth (LB) containing the control plasmid DNA exhibited colonial growth in the presence of ampicillin and lawn growth in the absence of it. For the broth with no plasmids, it demonstrated no growth at all with ampicillin and lawn growth without it. And the LB containing lux plasmid displayed colonial growth with ampicillin and lawn growth without.
1 Introduction
DNA and RNA are nucleic acids found within cells responsible for storing the genetic code for the development, growth and survival of organisms (Scherrer, 2018). DNA stores the original genetic code necessary for all living organisms to develop and survive. The mechanism for a gene to generate RNA and protein occurs through a process called gene expression. This development of RNA and proteins occurs almost simultaneously in a two step process labeled transcription and translation (Clark et al., 2018). In order to understand and employ the instructions for developing the organism, DNA is copied, or transcribed, into RNA so that the genetic information could then be converted, or translated, into proteins. As proteins, the information coding for the functional, structural, and biochemical components of the cell could be determined and expressed. This is the process by which enzymes read the genetic code to determine which and how many proteins to create in the organism. These biological processes occur in eukaryotes as well as prokaryotes. During transcription, the first step of the process, an RNA molecule is arranged from the DNA’s “template”. Upon exiting the nucleus, a mature RNA can be translated into a polypeptide sequence in the process of transcription (Alberte et al., 2012). These mechanisms take place in and are managed by the cell but biologists have found ways in which it can be manipulated, especially in prokaryotes. The field of study in which they do so is known as biotechnology. Biotechnology is the genetic manipulation or modification of biological processes for a specific purpose and brings about useful technological advances (Alberte et al., 2012). For instance, in healthcare, biotechnology has allowed for the production of hormones such as insulin, vaccines for the prevention of certain diseases, and antibiotics like penicillin and ampicillin.
In prokaryotes, DNA is found as circular molecules called plasmids. They carry genes which can aid in bacterial survival when expressed (Raven et al., 2011) As an illustration, some plasmids can have genes that code for antibiotic resistance. Bacterium with this gene can grow and multiply in the presence of antibiotics. This experiment is performed to form (Edwards, 2015) (Salari, 2012)Escherichia coli (E. coli) that are resistant to ampicillin through the addition of two plasmids, control plasmid pUC18 and plasmid lux DNA. Plasmid pUC18 containing the ampicillin resistance gene and plasmid lux containing the ampicillin resistant gene and a lux operon, giving bioluminescent features to the cells. The three conditions which need to be reached in order for transformation to be successful includes, a host for which DNA can be inserted, a measure for which the DNA can be transmitted into the host cell, and a way to identify the transformed cells (Alberte et al., 2012). Judging by the knowledge of how bacteria react to antibiotics and how transformation occurs, it is hypothesized that the successfully transformed bacteria will ampicillin resistance and grow, while those without the plasmid DNA will not.
2 Methods
Prior to the start of the experiment, the bacterial cells were treated with Calcium Chloride by the addition of 590 L of the solution into a test tube containing 50 L of E. coli, both of which were placed inside an ice bath. The solution was mixed and then placed on ice for at least 10 minutes, resulting in having competent cells capable of taking up DNA.
For the first portion of the experiment, the competent cells were incubated with one of two plasmid DNAs, plasmid lux or a control plasmid (pUC18), of which each group was assigned one of the two. It was done so by labeling small Eppendorf tubes, “C” for the control plasmid DNA or “lux” for the plasmid lux DNA. Both were placed in ice baths and 5 L, of either the control plasmid or the lux plasmid, were added into their respective tubes. 70 L of the competent cells treated prior to this procedure were then added to each of the tubes, mixed, and placed on ice for 15 minutes. In between the 15-minute window, a no plasmid tube was assigned to each of the groups containing 35 L of competent cells, to later serve as an illustration of how normal growth is supposed to look. Once the 15 minutes passed, the cells were Heat Shocked by being transferred to a water bath of 37 C for 5 minutes. Proceeding the 5 minutes, 275 L of nutrient broth were added to the control and lux tubes, 150 L to the no plasmid tube, and then incubated at 37 C for 45 minutes.
Cells that had taken up the plasmid DNA were selected for the second portion of the experiment by observing their growth on a plate containing ampicillin. Each group was given three agar plates to dispense and evenly spread 130 L mixed bacteria from the control tube, and 130 L bacteria suspension from the “lux” tube. Cells from the tubes containing no plasmids were placed onto the remaining two plates. The lids were re-placed on the plates, and they were left in room temperature for about 10 minutes for the purpose of the liquid being absorbed. The plates were then inverted and incubated at 37 C.
3 Results
Table 1
Treatment Observed Growth Type Bioluminescence
(yes or no) Reasoning
LBc Lawn Growth No Control & has plasmid/ doesn’t have lux operon
LB/Ampc Colonial Growth No Has ampicillin & plasmid resistance
LBNP Lawn Growth No No plasmid, no transformation
LB/AmpNP No Growth No No plasmid, no transformation
LB/Amplux Colonial Growth Yes Ampicillin resistance & lux operon
LBlux Lawn Growth Yes Ampicillin resistance & lux operon
Table 1 demonstrates the different treatments, whether or not there was growth, and whether or not it displayed Bioluminescence.
The observed growth type of the LB Broth with the control plasmid and no ampicillin, LBc, was lawn growth and same broth with ampicillin, LB/Ampc, displayed colonial growth. Lawn growth is illustrated as the bacteria covering the surface of the plate, evenly spread out. On the other hand, colonial growth shows up as spots or circles of bacteria congregated on different areas of the spot plate.
The LB Broth with no plasmids, LBNP, exhibited lawn grown with no ampicillin and no growth at all in the presence of ampicillin, LB/AmpNP. And the LB Broth with the lux plasmid, LBlux, displayed lawn growth without the ampicillin and colonial growth with ampicillin, LB/Amplux. All of the LB broths displayed no bioluminescence except for the two that contained lux plasmid. LB/Ampc, with the lawn growth, displayed 30 colonies and LB/Amplux, 40 colonies.
4 Discussion
As shown in Table 1, all of the plates exhibited some form of growth except for the LB Broth with no plasmid in ampicillin. This plate served as a negative control, as it displayed what a “normal” situation would look like in which the E. coli would be unable to grow due to the antibiotic. The LB Broth with no plasmid and no ampicillin served as a positive control because it demonstrated the normal growth pattern of the bacteria in the absence of antibiotics. The LBc and LBlux both illustrated lawn growth without the ampicillin. While with the ampicillin, they both presented colonial growth, LB/Amplux with the highest amount, 40 colonies, and LB/Ampc with 30.
The cells transformed with pUC18 and plasmid lux grew in the presence of ampicillin due to the ampicillin-resistance gene acquired from the plasmids, allowing them to endure the ampicillin from the agar plate and grow naturally. The Agar served as a solid growth medium in which the transformation efficiency could be tested while the Lysogeny (LB) Broth served as a liquid growth medium providing the bacteria with the nutrition to grow. The plasmid served as a vector for the transformation of the gene in the E. coli and the Ampicillin tested the efficiency of transformation of the ampicillin-resistance gene.
The phenotype of the transformed colonies indicated which cases of transformation were successful. That is to say that, the growth of colonies in response to the ampicillin indicated which bacteria successfully absorbed the plasmid while those displaying lawn growth indicated a normal growth pattern. The LB/Amplux plate, the plate of broth containing the lux plasmid and ampicillin, served to be the most successful transformation, with 40 colonies spotted. An enzyme, Luciferase, is produced by cells transformed with plasmid lux and not produced by the cells transformed by pUC18. Luciferase is the enzyme that catalyzes the light-emitting reaction, causing the bioluminescence in those cells containing the plasmid lux.
In nature, DNA uptake by different organisms can have its benefits as well as its disadvantages (Alberte et al., 2012). It could be beneficial in instances like this one where the uptake of a particular plasmid created an antibiotic resistance. A genetic transformation might be maladaptive to a host organism in a situation where it inhibits the cell’s ability to grow and reproduce. There is also the situation in which the uptake of DNA by a particular organism might be advantageous to that particular organism but disruptive to the surrounding ecosystem. To demonstrate, in the case where a type of prey takes up DNA that allows it to be resistant to the predator, the prey might be saved, but the predator’s food source is then eliminated. This would result in the endangerment of the predator species and an overpopulation in the prey, causing an imbalance in the food chain.
In the final analysis, the results concurred with the predictions that the successfully transformed bacteria would exhibit growth and ampicillin resistance, while those without the plasmid DNA would not. The plasmid lux showed fewer colonies due to the fact that it is larger than the pUC18 plasmid and therefore, less efficient in being absorbed by the cells. But due to the successful transformation, the bacteria that acquired the ampicillin-resistance gene from either the pUC18 of the lux plasmid were able to grow in the ampicillin-rich agar plate. Overall, the experiment was very well structured and didn’t display any signs of error. If done again, it would be important to see the effects of the lux plasmid’s bioluminescence in the dark, to see the full effect.
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