Aerobic Respiration Produces the Most

¶ … aerobic respiration produces the most ATP? Describe how this process works including where the entering molecules came from, the cellular location and how the ATP is produced.

Although some ATP nucleotide is produced via substrate-level phosphorylation, the majority of ATP generated during aerobic respiration comes from oxidative phosphorylation within the mitochondrion. The process of oxidative phosphorylation is driven by ATP synthase through utilization of energy in the form of proton motive force. The electrochemical gradient of proton motive force is generated through the electron transport chain, wherein electron donors, such as NADH, facilitate the transfer of protons across the inner mitochondrial membrane. This proton gradient then crosses back through the membrane, where its energy is utilized by the ATP synthase enzyme to phosphorylate ADP and generate ATP. The electron donors used in this process are created through the Kreb's Cycle.

Compare and contrast the processes of DNA replication in the cell vs. In a Polymerase Chain Reaction.

DNA replication in the cell is a highly regulated process that involves the single duplication of the cellular genome. Replication begins at designate sites within the genome, known as origins, where the initial unwinding of DNA occurs. Quality control mechanisms within the cell help ensure that aberrant incorporation of nucleotides and mutations are corrected, promoting a high level of fidelity during the replication process. Cellular replication is driven by DNA polymerases, and the replication process is highly controlled by cellular cycles and their associated cyclins and cyclin-dependent kinases.

Polymerase Chain Reaction (PCR), however, is an in vitro process whereby segments of DNA, and not the whole genome, are amplified exponentially within a test tube. The initiation of replication in PCR requires an initial primer fragment that associates with the DNA template to be replication. Since there are often no cellular quality control enzymes within the PCR reaction, the fidelity of the amplification process can vary depending on the polymerase being utilized. Given the high temperatures required for denaturing and annealing, polymerases from thermophilic bacteria are typically used.

3. The tsr gene in E. coli encodes a receptor that senses nutrients and allows the bacteria to find food sources. Trace the creation of the TSR protein starting with the gene in the genome and ending with the protein. (Include enzymes and molecules involved in the processes.)

The generation of the protein begins with the creation of its cognate mRNA. RNA is transcribed from the DNA associated with the protein by a RNA polymerase. RNA transcription involves pre-initiation, initation, elongation, and termination stages, wherein a wide-variety of proteins and protein complexes are involved. The mRNA, along with ribosomal components, are targeted to the bacterial membrane where co-translation integration of the receptor occurs. Translation requires the template mRNA, amino-acyl tRNAs, and amino acids, in addition to two ribosomal subunits (small and large), an energy source, and translation initiation factors. Co-translation folding within the membrane occurs as the polypeptide chain of the receptor collapses into its native fold. In prokaryotic systems, transcription and translation are often coupled.

4. Why are prokaryotes able to react to their environment by producing proteins much more quickly than eukaryotes? (This should be a complete analysis including multiple differences between proks and euks.)

Prokaryotes can react to their environment more quickly than eukaryotes because the transcription and translation processes are coupled and inherently possess fewer processing steps. In eukaryotes, transcription occurs within the nucleus, where the nascent RNA then undergoes several processing and splicing steps before translocation to the cytoplasm. Within the eukaryotic cytoplasm, the mRNA may be translated or it may be targeted to the ER for translation and subsequent secretion.

In prokaryotes, however, there is no nucleus and the transcription and translation processes are coupled. There is typically no processing of transcribed RNA and there is concomitant transcription and translation of mRNA. It is also possible that the polyploidy state of many prokaryotes during the continuous replication process can result in multiple copies of specific genes.

5. How would you determine whether or not the unknown bacteria in your patient's blood sample is Shigella flexneri? Describe the steps of your chosen technique including how the unknown bacteria would be identified.

One way to identify if the unknown bacteria within a patient's blood is S. flexneri is through isolation of colonies, morphological analysis and appearance, biochemical assays including urease, mannitol and oxidase tests, and agglutination with anti-sera. Based on the results of these assays, S. flexneri can often be identified, although additional kits may be required. The simplest way, however, may be the novel approach through multiplex PCR (mRPC). It is possible to identify Shigella species through mPCR techniques by identifying pathogenicity islands associated with Shigella and S. flexneri.

6. How could you create a corn plant that would express the human protein fibrin? (You need to include techniques, steps, enzymes, etc.)

In order to create a corn plant that would express the human protein fibrin, scientists would first need to incorporate the human fibrin gene within the corn plant genome. The incorporated human gene would require regulation and promoter sequences that would function within the plant cell. Proper splicing sequences would also be required or removal of the introns altogether.

The delivery of transgenes into the corn plant could be accomplished through electroporation into corn protoplasts followed by stochastic recombination into the plant genome. Electroporation is thought to generate transient pores within the plasmalemma and facilitate transfer of the DNA inside the plant cell. Integration would subsequently be enriched or selected for in order to identify plants which have successfully incorporated the desired transgene.

7. If you were the lab technician hired by the Jerry Springer show, how could you determine which of the 4 possible men was the baby's biological father? (Include the theory and techniques involved in the identification.)

The identification of the baby's biological father can be accomplished through standard paternity testing. Paternity testing is a "genetic fingerprinting" method that involves PCR amplification of specific regions of the father's and child's DNA, followed by restriction enzyme digestion. Since all humans possess polymorphisms, they thus have restriction fragment length polymorphisms as a result of differing restriction sites within the DNA. Since the fragment lengths of the digested DNA is extremely variable depending on the individual restriction sites, the likelihood of having similar length fragments amongst individuals is low unless they are blood related. Therefore, half of the fragments of the father will be identical to the child's DNA fragments, since the child will possess half of the father's DNA. The other men will possess restriction fragments of varied lengths that are dissimilar to the child's fragments.

8. Imagine that you are planning to treat a patient with the antibiotic Kanamycin for his Staphylococcus aureus infection. Explain how you would determine both: A) the Minimum Inhibitory Concentration of Kanamycin for this infection and, B) the Therapeutic Index of Kanamycin. Include an explanation of why this information is important.

The minimum inhibitory concentration of kanamycin can be determined through standard agar dilution techniques or disk diffusion methods, wherein the concentration of antibiotics necessary for inhibition can be determined using S. aureus. The therapeutic index of kanamycin is the ratio between the median lethal dose and the effective dose. Both the MD50 and the ED50 are known values, since they are based on the population testing (of animals). It is important to know these values so that the physician can determine the appropriate amount of antibiotic to be administered, without placing the patient danger but also maintaining its effectiveness against the patient's S. aureus infection.