Sp Lab Report

With all living organisms, a process known as cell respiration is integral in order to provide the body with an essential form of energy, adenosine triphosphate (ATP). Oxygen, although an essential part of this process, can form reactants from colliding with electrons associated with carrier molecules. (pb101.rcsb.org, 2017). Hydrogen peroxide is an integral product of this reaction but is known to impose negative effects on the body if high levels are introduced. Explicitly, this reaction is caused “If oxygen runs into (one of these) carrier molecules, the electron may be accidentally transferred to it. This converts oxygen into dangerous compounds such as superoxide radicals and hydrogen peroxide, which can attack the delicate sulphur atoms and metal ions in proteins.” (pdbh101.rcb.org, 2017). Research has suggested that the hydrogen peroxide can be converted into hydroxyl radicals, known to mutate DNA, which can potentially cause bodily harm due to DNA’s role in the synthesis of proteins. These radicals can cause detrimental effects on the human body, and studies have suggested a link to ageing. Due to the harmful effects of these H2o2, it is important that the body finds a way to dispose of hydrogen peroxide before concentrations are too great.

There of the dishes turned out as expected in this experiment. Our group expected there to be growth in the LB -pGLO dish as the bacteria were not exposed to the antibiotic ampicillin. Furthermore, our group also expected to see inhibited bacterial growth in the LB/amp +pGLO dish as there was ampicillin in the dish, but some of the bacteria were immune as they possessed immunity to the ampicillin. Moreover, our group expected that there would be no bacterial growth in the LB/amp -pGLO dish, as the bacteria were exposed to ampicillin and were not immune. However, the final dish, LB/amp/arbo +pGLO, did not turn out as expected. While it was expected to allow for inhibited bacterial growth and the bacteria to become florescent,

The purpose of this study was to see whether E. Coli cells would engage in the pGLO plasmid and glow in the presence of four control environmental factors which are arabinose sugar, bacteria, the antibiotic ampicillin, LB nutrient broth and pGLO plasmid DNA. This was tested using four plates, all the plates had E. Coli cells and different environmental factors. The founding was that E. Coli will only fluoresce when bacteria, pGLO plasmid DNA, the antibiotic ampicillin, and LB nutrient broth are present. The result did not support the hypothesis because it stated that, E. coli cells that are exposed to the pGLO plasmid would engage in the plasmid and glow only if the arabinose sugar is present.

This method is called reduction-oxidation. Reduction-oxidation was tested on the most treacherous antibiotic-resistant infections such as: Staphyloccus aureus, Klebsiella pneumoniae, Salmonella typhimurium, and Escherichia coli. This resulted with no reaction, yet an improvement on cellular reproduction. On the other hand, Reduction-oxidation was successful in terminating weaker antibiotic-resistant bacteria (Nagpal,

These superoxides may be dangerous because they alter the structure of iron and protein via reduction. They may also undergo dismutation to form hydrogen peroxide which, in turn, gives rise to hydroxyl radicals, the most reactive ROS (Gulumian and Van Wyk, 1987; Agarwal et al., 2005). Hydrogen peroxide is not a free radical but its neutral charge allows it to pass through cell membranes and so this makes it very dangerous (Kurutas, 2015). Other internal or endogenous sources for these free radicals are inflammation, xanthine oxidase, peroxisomes, phagocytosis, exercise and ischaemia. Exogenous factors which lead to the development of these ROS include smoking, ozone, environmental pollutants, radiation, pesticides and drugs (Lobo et al.,

These mutations, no matter what process that has led to their occurrence, block the action of antibiotics by interfering with their mechanism of action (1). Currently, antibiotics attack bacteria through one of two mechanisms. In both mechanisms the antibiotic enters the microbe and interferes with production of the components needed to form new bacterial cells. Some antibiotics act on the cell membrane, causing increased permeability and leakage of cell contents. Other antibiotics interfere with protein synthesis in cells. They block one or more of the steps involved in the transformation of nucleic acids into proteins.

They can also interfere with how proton pumps in the bacteria cell membrane work, decreasing membrane integrity (Raut & Karuppayil, 2014, p. 252)

The initial stages of this bactericidal activity is thought to arise due to the peptides anchoring themselves to the bacterial membrane via the DAB amino acids [197, 198]. While the precise mode of action following this behavior is still unclear, it has been established that the polypeptide ring is responsible for creating an increase in permeability of the bacterial membrane [25]. Similarly, the nonapeptide analogue of PMB1 also has this characteristic [199]. It has been proposed that the observed permeabilization is caused more specifically by membrane insertion of the polypeptide ring and fatty acid tail, causing disruption in theresulting in bilayer disruption and an outflow of intercellular components, leading tofollowed by cell death

Overtime, the evolution of bacteria has been utilized as a tool to produce medicines such as insulin and even varios growth hormones. Transformational success of bacteria is mainly due to versatile genetic coding, enabling researchers to transfer DNA to numerous amounts of organisms. Studies have utilized quantitative analysis to design experiments and expose Escherichia coli, commonly known as E-Coli, to a plasmid surface obtaining genes that have the potential to resist Ampicillin, which is identified as an antibiotic. A substance known as Calcium chloride was used to eliminate repulsion that existed between the DNA and cell wall of the bacteria. The Heat shock method was utilized to give DNA a required energy boost to enter the bacteria

According to the results of the experiment located on Image 1 in the results section, the phenotype of the E Coli demonstrated a change when exposed to the plasmid pGlo. This conclusion disproved the hypothesis. All four agar plates were exposed to LB broth (Luria broth). This broth is the source of food and nutrients E Coli used to grow and divide. As demonstrated by Image 1, there was growth in the in the agar plates -LB, +LB/amp, and +LB/amp/arab. The substantial growth that was demonstrated in the -LB agar plate (estimated at 75-80% growth) was due to this was the standard E Coli without any ampicillin or pGlo present. Moreover, the agar plate for +LB/amp showed a margin of growth. The percentage of growth was estimated at a 45-50% growth and signified this in individual colonies. This plate contained the plasmid pGlo. This plasmid is coded with GFP protein, antibiotic resistance gene, and the araC gene (www.bio-rad.com). Even though the agar plate contained the antibiotic ampicillin which kills the E Coli bacteria, the E Coli still grew because the plasmid pGLO was present. This growth demonstrated that the E Coli bacteria absorbed the pGLO plasmid and revealed a change in the E Coli’s phenotype. There was a margin of growth because the E Coli was subjected to the transformation solution (CaCl2) and the heat shock. A

antioxidant enzymes including small-molecule-weight antioxidants depends on cellular redox environment as it is a delicate process to regulate the two. ROS are responsible to regulate several physiological actions such as the ability to mediate and relate signal transduction from membrane receptors, At low concentrations, ROS are involved in regulating several physiological actions, including their ability to mediate relate signal transduction from membrane receptors, thus aiding the stimulation of several proteins and enzymes (1,2). Conversely, accumulation of extra intracellular ROS lead to oxidative stress, in turn will impair cellular membranes, promoting mitochondrial injury and cell death, which adversely impacting upon cell function and survival 3,5)

To increase their effectiveness, many organisms resort to producing molecules called virulence factors. These molecules prove to be beneficial to the pathogen by strengthening their pathogenic capabilities. Through many examinations and dissections performed on S.aureus, it was revealed that that bacteria itself was home to a wide range of virulence factors. Being the most common, the pathogen is comprised of surface adhesions whose main job is to regulate the attachment of the bacteria to its target tissues(4b). This in turn helps guarantee the bacteria a better chance of invading the tissue and causing an infection. In addition, another important factor is the polysaccharide capsule(4c). Even though many variations of the capsule have been reported, type 5 and 8 have been deemed to play a more

Similar data have been reported previously by Elkady and Ibrahim [36] and Kutanis et al. [40]. Meanwhile, pretreatment of HMB prior to IR exposure increased the antioxidant levels and decreased the levels of MDA when compared with IR-exposed rats. These results showed that HMB attenuated IR-induced lipid peroxidation and that was compatible with the study of Rao et al. [22] who suggested that the decrease in MDA concentration could be due to the ability of HMB to scavenge secondary reactive radicals or to prevent the formation of superoxide or hydrogen peroxide in response to IR exposure. It was stated that HMB is a potent ONOO_ scavenger that can protect the cells against peroxy nitrite-induced diseases [41]. Along these lines, the reaction between O2·- and NO may be diminished by the activity of HMB, whereas the enzyme-catalyzed reaction by SOD would debilitate in this manner thus, the SOD can be maintained. It was reported that HMB can reduce hydroxyl radical and presumably repress the formation of H2O2 in the reaction chain, which may at last prompt the maintaining of intracellular GSH levels [18, 42, 43]. Antioxidant, activity of HMB in our investigation, may be ascribed to the phenolic nature of HMB which could have prompted free radicals scavenging [18]. However, the

Similarly, high level of attachment was also seen in one of the E.coli O157:H7 strains. Although the fluorescence microplate assay values for the Salmonella strains (even for the one categorized as strongly adherent) were relatively low compared to adherent strains of L. monocytogenes and E. coli, we selected the most adherent strain among the Salmonella to continue our microplate lethality assays with sanitizers on biofilms of strongly adherent strains from these 3 groups of pathogens (Fig. 10). These data suggest that Salmonella spp. may be weak at retention of 5,6-CFDA, or possibly that biofilms may exude a protective coating that limits 5,6-CFDA entry by diffusion. This proposition that bacterial species might have varied levels of fluorescence labeling or retention capability has been studied before (Drevets & Elliott, 1995). Another possible reason that can be argued for low RFU values with Salmonella is the incubation time of 15 minutes with the substrate, which may not be enough for the 5,6-CFDA substrate to enter the cells and get hydrolyzed to provide fluorescence.

Moreover, selenium has also been found to trigger the generation of ROS, with both elements capable of reacting with intracellular thiols and forming intermediates that cause oxidative stress as a consequence of the formation of superoxide radicals [21, 22]. Nanoparticles can contribute to functional damages of cell membrane or wall by disrupting the integrity of these important envelopes [23].