Throughout the urea cycle, the amino acid, arginine, is changes into ornithine- this is another amino acid when hydrated, that is when water was added. During this reaction, urea is the product formed (Nelson and Cox 2008). Figure 1 shows the urea cycle, occurs specifically in the mitochondria and cytosol in the liver. (Nelson and M.Cox 2008). Urea is made in the liver by means of enzymes in the urea cycle.
Following are the tests used for the identification of bacterial species based on the differences in the biochemical activities of different bacteria. Beta glucouronidase test is used for the identification of Escherichia coli. An enzyme is produced by E.coli which is beta D glucouronidase. Beta d glucouronidase in turn hydrolyzes beta d glucopyranosid uronic derivatives to aglycons and D glucuronic acid. Bile solubility test is used in laboratory for differentiation of alpha hemolytic Streptococci from Streptococcus pneumoniae.
Differential organisms require different nutrients which show it is fairly easy to isolate and identify organisms by using selective media. Differentiation media are also used by taking advantage of whether an organism produces acid or not. MacConkey agar is an example of a differential media which will enable the organism to express a colour depending on whether it produces acid. This agar contains a pH indicator which can provide more information on the type of bacteria that is growing on the agar. Method A more detailed method is provided in the 280.201 Industrial Microbiology lab manual.
2. You have been asked to set up a dilution series, and then use spread plates to determine the viable cell count. Why is it necessary to use a dilution series when isolating bacteria from a biological sample using spread plating? [5 MARKS] It is vital to use a dilution series to reduce the concentration of the original biological sample so it is easier to count the number of isolated colonies which are present on the spread plate. The diluted samples will have a workable number of colonies on the spread plate which can then be used to calculate the number of bacteria which were present in the original undiluted sample.
• Serine, threonine and cysteine proteases use a nucleophilic residue (usually in a catalytic triad). That residue performs a nucleophilic attack to covalently link the protease to the substrate protein, releasing the first half of the product. This covalent acyl-enzyme intermediate is then hydrolysed by activated water to complete catalysis by releasing the second half of the product and regenerating the free enzyme. A comparison of the two hydrolytic mechanisms used for proteolysis. enzyme is shown in black, substrate protein in red and water in blue.The top panel shows 1-step hydrolysis where the enzyme uses an acid to polarise water which then hydrolyses the substrate.
The secondary antibody would the overlap the primary antibody by binding to it. The secondary antibody (anti-rabbit HRP labelled) used is conjugated with an enzyme Horse radish peroxidase which then binds to its substrate tetramethyl-benzidine (TMB) to produce a blue color indicating the presence of lysozyme. SDS-PAGE gel electrophoresis is a process which separates proteins based on molecular weight. The purpose of this method is to separate out the lysozyme by its weight. The weight is known to be 14.6Da.
A plaque assay is a modification of a bacteriophage assay and is used to estimate the titer (concentration of a solution) of a phage stock. A plaque assay contains a virus combined with bacterial cells on the surface of an agar plate. The agar plate inhibits the spread of viral progeny to neighboring bacterial cells which results in plaque formation (lysis of bacterial cell). The purpose of this lab was to perform a bacteriophage assay and to calculate the original stock titer of the phage stock using the known set of dilutions and direct plaque count. The hypothesis for this experiment was that plaques will form on all the agar plates within a range count of 30 to 300.
Lactobacilli adhesins can be broadly classified according to their targets in the intestinal mucosa (i.e. mucus, extracellular matrix), based on their localization in the bacterial surface (i.e. surface layer proteins) and/or to the way how they are anchored to bacterial surface (i.e. sortase-dependent proteins, N/C terminal anchored). Extracellular matrix (ECM) comprises of variety of proteins like collagen, fibronectin, laminin along with mucins.
Bacteria taking up foreign DNA is known as transformation. Transformation implies uptake in bacterial, yeast or plant cell DNA while transfection is the term used in reference of mammalian uptake. Chemical transformation, electroporation or particle bombardment is the typical method of construct into a host cell. Conjugation: The easiest illustration is to consider this as a version of bacterial sex. In conjugation the two bacterial cells connect, and the male donates a piece of DNA to the female.
There are enzymes located on the intestinal brush border known as ferrireductases which reduce ferric iron to the more soluble ferrous state. Once reduced, the ferrous iron is then transported across the mucosal cells by the transporter protein divalent metal cation transporter 1
The link between bacteria and ulcers was then established. After a great deal of research, it was revealed that in individuals that were infected with Helicobacter Pylori, antibodies were found in the bloodstream. Helicobacter Pylori are able to attach to cells as they go through the mucous layer of the stomach. When the bacterium enters the stomach, an enzyme called urease converts the stomach cells chemical urea into ammonia and carbon dioxide. The enzyme urease tends to trigger inflammation.
The repressor is a regulatory protein that binds to the operator and blocks transcription of the genes of an operon. Inducers bind to the repressors and they also regulate gene expression. In the process of identifying the three strains of E.coli, ONPG (ortho-nitrophenyl b-D galactoside) was used as an indicator. ONPG is a substrate that can detect B-galactosidase, and when it does, it turns yellow. Sarkosyl was also a detergent used in the lab to lyse open cells.
Mannitol high salt testing is done in order to determine if the bacteria is salt tolerant and can ferment mannitol. Catalase activity test establishes whether the bacterium produces the enzyme catalase. The eosin methylene blue test or EMB, inhibits the growth of gram positive bacteria and tests whether or not gram negative bacteria can ferment lactose. Lactose fermentation testing is done to see if the bacterium is capable of fermenting sugar by testing for acid and gas production. These are the possible tests that are needed in order to identify unknown
Conversely, Klebsiella pneumoniae and Escherichia coli are both coliforms, which are able to ferment lactose. Of the Enterobacteriaceae family, there are genera that are in the normal human flora. Some species such as K. pneumoniae and E. coli are opportunistic pathogens which can capitalize on weakened host defenses and cause food poisoning (Baron, 1996). S. enterica secrete proteins that help aid in intracellular invasion and proliferation (Hensel, 2009). K. pneumoniae is a part of the normal human mouth, skin, and intestine flora, but can wreak havoc if inhaled (Ryan,