FRAGMENT OF DNA: E. COLI Sample

Introduction 

The cellular metabolism has been overly investigated for more information of the cellular functions that would allow understanding some cellular organisms to adapt to various environmental changes within levels of variations. It is important to analyze such reconstruction of metabolic activities of bacteria Escherichia coli and Staphylococcus aureus because they are both bacteria that could easily infect man because they are widely distributed in the environment.

Furthermore, it is important to identify their metabolic cores of these organisms as it would represent connected sets of reactions that these organisms used to test their environments. The study would be studying the fragment of DNA to be associated with the cellular metabolic activities of the mentioned organisms. The DNA fragments of Escherichia coli and Staphylococcus aureus would be subjected to further comparison and differences giving further recognition on their respective uniqueness as cellular organism while supporting the notion that core reactions may represent integrators of metabolic activities.

 Aims & objectives

The aim of this study is reproducing and identifying the independent strains with the DNA fragments of the Escherichia coli and Staphylococcus aureus.

• The experimental objectives are to
• conduct a culturing of Escherichia coli
• Apply PCR-based amplification of the Escherichia coli DNA
• Apply Electrophoretis separation of the Escherichia coli DNA
• reproduce independent strains with the DNA fragments of Escherichia coli andStaphylococcus aureus
• Identify the same DNA fragment (Reed &Palsson, 2004).

Hypothesis

1. H1: There is a significant difference between reproducing and identifying the independent strains with the DNA fragments of the Escherichia coli and Staphylococcus aureus.
2. H2: There is no significant difference between reproducing and identifying the independent strains with the DNA fragments of the Escherichia coli and Staphylococcus aureus.

Background of the project and context

Identification of the Bacteria

Escherichia coli or widely known E. coli domain is a bacteria gram-negative within a large and diverse group. E. coli 2-3 microns long rod shaped is commonly located in the large intestine beneficial and harmless 0.1% of the gut flora.

Most E. coli strains are beneficial preventing pathogenic bacteria in the intestine and assists production of vitamin K though the rest are disturbingly harmful that may cause ailments like diarrhea, tract infections, and pneumonia. Because the bacteria could easily adapt to any environment and also reproduce rapidly, E. coli can be found in both land and water making even drinking water contaminated (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004).

There are different kinds of E. coli enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), enterohemorrhagic (EHEC), enteroaggregative (EAggEC) and yet the STEC creates the Shiga toxin that causes several diseases. The Shiga toxin-producing E. coli O104:H4 had already been the root cause of massive damage due to its unexpected outburst at Germany in 2011. The acknowledged STEC was located mostly at North America is E. coli O157:H7 (Edwards &Palsson, 2000). 

Most E. coli outbreaks are from E. coli O157. The infection would actually begin when the person or any animal would have swallowed STEC (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004). This means that the person or animal was able to eat a microscopic amount of human or animal faeces. Though it is unnoticed, most of the E. coli O157 happens most of the time. Exposures ranged from consumption of contaminated food, consumption of unpasteurized milk, consumption of water that was never disinfected, contact with cattle and other animals without washing the hands and having contact with the faeces of infected people. Unfortunately, E. coli o157 can also be detected in some foods that could produce high infection. Such foods are the raw milk, incomplete pasteurized apple cider, and any by-products from raw milk. Other contacts are visibly obvious like working with cows and dairy products, taking care of the sick, baby sitting with contact to changing diapers (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004). Some people get infected when exposed to public water sources like drinking water from the sea, the lake, touching the environment in petting zoos and other animal exhibits or by feeding the animals without washing the hands and after using the toilet.

Staphylococcus aureus round or coccus shaped gram-positive bacteria, which appears purple. Commonly known as part of the natural flora located mostly on the skin.

Yellow colony arranged in group’s bacteria size of 0.5-1.5u on nutrient agar medium.

The staph.aureus mostly being beta-hemolytic strains grow either aerobically or anaerobic outstanding on blood ager medium.

Infectious causing staph.aureus can variety from little skin problem such as pimples to meningitis or toxic shock syndrome diseases. The spread causing the infection can be by direct contact of skin to skin or mucous penetration. Other types are such as staphylococcusepidermidis, lugdunensisor saprophyticus.

DNA Sequencing

This is the recognizing the placement of the nucleotides within the molecule of the Escherichia coli DNA molecule. Current method within technology had been used within the four bases of processing the strand of DNA. These are adenine (A), guanine (G), cytosine (C), and thymine (T). In some cases the purine (A+G) and pyrimidine (T+C) is discovered to be chemically modified in bacteria. (Randall K. Holmes and Michael G. Jobling.).

The rapid DNA sequencing would accelerate the biological and medical research about Escherichia coli due to the discovery of how to manage its actions of infections. There is a need to know the DNA sequences due to the indispensable basic biological research that would apply the diagnostic, biotechnology and forensic aspects of the systems of biology. Though there is a sequence to complete the DNA of E. coli, the genomes of the bacteria would have a complete DNA sequence. The improved automated sequencing method has now proven to place an accurate sequencing up to a maximum of 700 – 800 base pairs within length. It is also very possible to have full sequences of larger genes within the whole genomes from applications of either Primer Walking or Shotgun sequencing.

These two applications have greatly helped with the reconstruction of the sequencing of DNA especially for medical research relating to curative measures of common diseases. With Primer Walking, there is a larger portion of the gene being sequences with the Sanger method. The primers are to be generated from a reliable segment of sequence until the sequencing continues to a portion that the gene was out of range during the original reactions. The Shotgun sequencing meanwhile involves the cutting of the DNA segment into more manageable sizes of fragments to be sequenced and arranged within the pieces based on the overlapping sequences (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004). Such technique allows an easier approach from the help of computer software in arranging the overlapping pieces.

• Organisms E. coli
• StrainO157:H7
• Gene 1-561
• Plasmid pO157
• Locus AF074613
• Size (bp) 92077
• Locus/Protein_AAC70069.1
• Amino acid 186

Physiology of the system

Escherichia coli are bacteria very much in quantity in the intestines of humans and animals. There are thousands of strains identified in the intestines but only E. coli O157:H7 was seen dangerous to people because of its natural occurrence to produce toxins. The outbreak of contaminated hamburgers in 1982 in the United States was the introduction of the E. coli o157:H7 as there were many who fell gravely ill after eating the hamburgers. Soon, the medical research was able to also find other deadly strains of E. coli O104:H4 which were also part of the bacteria that had evolved in the 1960s to which shockingly disturbed microbiology for its swapping of gene with another bacterium called the Shigella (Lobersli, Haugum&Lindstedt, 2012). Currently, the E. coli O157:H7 had already contaminated ground beef. The butchering process of meat needs to be highly checked for cleanliness. It is present in the intestines of the slaughtered animal.

It can get into the meat as it is ground into hamburger (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004).
It was the summer of 2011 in Germany when the mysterious E. coli strain was able to infect almost 51 people making them lose their lives in the process. There was one Swedish and American who also got infected. The other 4,000 infected persons became terribly sick. The deadlier strain of E. coli, O104:H4 became viral that led to the numerous incidents (Lobersli, Haugum&Lindstedt, 2012). The reports claimed that the E. coli was able to elude the health officials when the health authorities announced to the people to refrain from eating cucumbers, tomatoes, lettuce and vegetable sprouts to have been claimed carriers of the bacteria (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004). It was a sensitive case of investigating which plant and place was highly contaminated. The investigation went on for months until they found the problem that was coming from sprouts growing in an organic farm of Bienenbuettel, northern part of Germany. The health officials in Germany immediately tested the crops for E. coli presence and found that such sprouts were eaten raw and did carried the said bacteria molecules. After 2 months from the outbreak, the European Food Safety Authority claimed a massive contamination of the fenugreek seeds from Egypt (Wiback SJ, Famili, Greenberg &Palsson, 2004).
In the United States, the highest rate was due to the initial condition of bloody diarrhoea. There should be proper food handling in order to prevent further exposure to E. coli especially of ground meats and other food that are not properly cooked. There is a need of refrigerating the meat as soon as it was bought (Wiback, Famili, Greenberg &Palsson, 2004). Thawing of frozen meat should not be done in the counter but in the refrigerator. Towels and face clothes should be washed separately with hot water and even bleached.

Critical review

The DNA restructuring and further research about the E. coli DNA was done through the use of microscopy wherein there was a conjunction of the staining of the visible material while (2) having acculturation of the bacteria to a large extent of observing the mass of bacteria production. The single bacterium was placed into binary fission in order to produce the characteristic mass of the identical bacteria (Schilling, Letscher&Palsson, 2000). Such colony was also tested to be genetically pure placing consistency on the shape, color, border and even elevation of the species of bacteria.  Broth culture was also used to create a tubing of the regional growth of the bacteria allowing the pellicle to surface within the fluid appearing to have stick within the bottom of the tube (Schilling, Letscher&Palsson, 2000). Nonetheless, the examination shows the bacteria’s oxygen requirements allowing the separation and isolation of the different bacteria types within the required nutrients of the broth.

Methodology

The first method is culturing would provide further information of the Escherichia coli by growing them on media containing nutrients. Because the bacteria are vastly detected in any place, the Escherichia coli may have been within a mixture of different bacteria (Papp Pal & Hurst, 2004). Culturing then would allow isolating Escherichia coli after using solidified media. The individual cells, which are diving within the surface may not move away from each other comparing its locomotion in water. Nonetheless, after several replications, there would be a formation of visible colonies that would compose about tens of millions of cells coming from a single cell. The study would like to attest a portion of the colony within a transferred liquid medium (Schilling, Covert,Famili, Church & Edwards, 2002). Then is the PCR-based amplification of the Escherichia coli DNA. The PCR based amplification can be easily applied on the DNA of Escherichia coli so as to generate millions of copies of a required DNA sequence. In case of E coli DNA the process will involve for three stages namely heat denaturation of the DNA molecule carrying the target sequence, Primer annealing as well as extension. The next step happens to be Electrophoresis separation of the Escherichia coli DNA. This can be carried out by usage of electricity and different sized pores. The electric charge will help in pulling of DNA molecules on basis of positive and negative charge. The matrix with pores will separate the molecules based on size of DNA and the pores. In order to detect the similar fragments of DNA, there can be usage of southern blotting techniques. 

Other research have expounded the use of the presumptive identification of 

Escherichia coli O157:H7 which is possible within an individual, non multiplexed PCR once there is a reaction with the enterohemorrhagicE. coli (EHEC) eaeA gene (Reed ,Vo,Schilling&Palsson, 2003). Though this study would describe the development and even the evaluation of the sensitivity and specificity of a PCR-based 5′ nuclease assay for presumptively detecting E. coli DNA, there would be a specification of the eaeA-based 5′ nuclease assay system being compared to previous studies of all E. coli O157:H7 strains evaluated. Making previous research as credible references, there is a mirroring of the previously specificity of the PCR primers(Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004).

Previous researches had done the separation of double-stranded and even single-
stranded DNA that was separated during non-denaturing conditions. Somehow both agarose and polyacrylamide gels can be used for separation of single-stranded Escherichia coli DNA (Burgard, Nikolaev, Schilling, Maranas, 2004). The size of the Escherichia coli DNA molecule was revealed when Escherichia coli DNA was compared to the band of interest with the bands of an appropriate Thermo Scientific DNA ladder on the same gel (Gerdes, Scholle, Campbell, Balazsi&Ravasz, 2003. . Because the Escherichia coli are single-stranded molecules, there is the separation on polyacrylamide gels in the existence of strong denaturants (7-8 M urea or formamide) and at high temperature or in agarose gels at alkaline pH (protocol) (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004). The Electrophoresis conditions would actually change depending on the voltage and even the composition of the
electrophoresis buffer (Almaas, Kovács, Vicsek, Oltvai&Barabási, 2004).

Expected Outcomes &Specific Investigatory Approach

Following outcomes have been derived in the present report. The conduction of culturing for E coli bacteria led to isolation on solidified media. The PCR based amplification helped in generation of a millions of copies of the required DNA sequence. The Electrophoresis technique led to separation of Escherichia coli DNA followed by reproduction of independent strains on the culture medium.

With such expected results, the specific investigatory approach would dwell in both (1)research and (2) experimentation previous studies about the capability of the E. coli molecules are placed within the same procedures of (1) culturing of bacteria, (2) PCR-based amplification of the DNA and (3) electrophoretic separation of the DNA under the experimentation phase of the study. (Duarte, Herrgard&Palsson, 2004) claims that before such experimentation, there should be a prior research on the nature of the E. coli bacteria molecules considering that such bacteria has been widely known to be deadly due to the non-existence of symptoms but internal damage of the disease. Currently, there is an ongoing E. coli long-term evolution experiment which tracks the changes in genetic codes from 12 sets of populations in the asexual Escherichia coli bacteria (Burgard&Maranas, 2001). And with the outbreak in Germany and Europe, the experiment continues its inception of evolutionary adaptations within 12 populations by applying citric acid as carbon source within aerobic environment (Burgard, Vaidyraman&Maranas, 2001). The said long-term evolution experiment would intent to provide further evidences on similar experiments on the evolutionary biology of the evolution of phenotypic in genomic levels. The use of E. coli would allow further generations to study the bacteria even within a frozen and preserved state. (Lobersli.,Haugum&Lindstedt, 2012) reported that in 2012 , the research team of Lenski was able to gain the genomic analysis of the E. coli strain that shed light to some genetic basis of evolutionary strain especially as it was seen to have evolved and merged with other bacteria. The research used the sequence of the entire genomes within the twenty-nine clones isolated within the population’s history (Studier, Daegeln, Lenski, Maslov& Kim, 2009). The sequences were reconstructed within the phylogenetic history within 20,000 generations. The clones were potentiated within the research of concluding the two mutations of E. coli. The research result claimed that the pair was able to promote the gene and verified its capability to reproduce asexually in almost any type of environment making E. coli dangerous bacteria (Price, Reed &Palsson, 2004).

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