Nika Vafadari Week 3

Electronic Lab Notebook Week 3

Purpose

To evaluate a primary research article and prepare for peer discussion, two skills critical to the fields of biology and mathematics.

Preparation for jornal Club 1

Biological Terms

1. Northern Analysis: Also referred to as Northern blot analysis. Method that can be used to detect specific RNA molecules or sequences and analyze particular gene expression. It works by denaturing the RNA then separating the molecules by size through gel electrophoresis. Next it utilizes capillary action to transfer them to a blotting membrane. The membrane then identifies specific RNA sequences using a nucleic acid probe that has a radioactive label. Northern analysis is a useful method since it can compare the abundance of mRNA samples on one membrane.
2. Amino Acid Permease: Integral membrane proteins that play a role in the transfer of amino acids across the semi-permeable membrane of a cell, thus allowing amino acids to enter the cell.
3. Oligonucleotide: A molecule, more specifically a polynucleotide, that consists of a small number of nucleotides that can hybridize to specific RNA or DNA sequences.
4. Alpha-ketoglutarate: An intermediate in the Citric Acid Cycle, thus playing a critical role in the biosynthetic processes of a cell. It can be used as source for the production of both glutamate and glutamine, since it is a precursor for these molecules, and is referred to as a “nitrogen scavenger,” as seen in Wu et. al referenced below.
5. Glutamate: Negatively charged amino acid that is a precursor for glutamine. It is also a neurotransmitter that plays an essential role in normal brain function.
6. Intracellular Metabolite: Molecules, such as alpha-ketoglutarate, glutamate, and glutamine, that play a role in the various pathways found in the intracellular metabolism of a cell.
7. Intracellular Metabolism: Made up of various enzymatic reactions and consists of three main stages, which include the synthesis and breakdown of compounds, such as proteins, the conversion of molecules through the use of intermediate metabolites and interconversion of metabolites through various reactions, such as phosphorylation.
8. Acetaldehyde: Molecule that acts as an intermediate in the TCA cycle where it is converted to acetic acid then to carbon dioxide. Plays a role in the metabolism of alcohol.
9. Glutamine: The most abundant out of the twenty amino acids found in the body, therefore playing a critical in the biosynthesis of various proteins.
10. In vitro: Refers to a process or experiment that has been performed outside of an actual living organism in a culture dish or tube.

Outline

Main Result Presented in Paper

• The concentration of ammonia (not the flux of ammonia) acts as the key factor in the regulation of nitrogen metabolism.

Significance of Work

Determine that the concentration of ammonia acts as the regulating factor in the metabolism of nitrogen in Saccharomyces cerevisiae, thus supporting previous studies mentioned that came to the same conclusion. In addition, the study found a trend of increasing biomass concentration of the yeast with an increase in ammonia concentration, therefore this information could be used to effectively grow cultures of Saccharomyces cerevisiae and increase there size. Increased growth in Saccharomyces cerevisiae could prove beneficial when using them in the process of making beer and bread as discussed in class.

Limitations in Previous Studies that Led to the Performance of this Work

• Previous studies determined
  • Saccharomyces cerevisiae prefer ammonia as a source of nitrogen, as opposed to other options, such as proline and urea
  • The regulation of various components involved in nitrogen metabolism occurs at the stages of gene expression and enzymatic activity

• While studies done using continuous culture identified ammonia concentration as an influencing and key factor in nitrogen metabolism
  • the flux of ammonia was not controlled during the experiments since the concentration of external ammonia and assimilation rate of ammonia were found to vary within the cultures
  • thus raising the question of the significance of flux as the key factor, as opposed to ammonia concentration.

Methods Used

• Physiological Parameters
  • Grew S. cerevisiae SU32 in continuous cultures with a fixed concentration of glucose (100mM) and various concentrations of glucose (29, 44, 61, 66, 78, 90, 96, 114, and 118 mM) in order to explore the effects of ammonia on enzyme activities and gene expression
    • dilution rate of medium= 0.15 h-1
  • Biomass concentration was measured at fluctuating ammonia concentrations
    • identified ammonia concentration as a limiting factor to biomass since a trend of increasing biomass was observed with an increase in ammonia concentration (up to 61 mM)
    • identified an increase in biomass concentration, while residual ammonia concentration remained constant, when ammonia concentration increased from 29 to 61 mM
    • however, when ammonia concentration reached above 61 mM, biomass concentration remained constant, while residual ammonia concentration increased
    • glucose identified as limiting factor when ammonia concentration reached above 61 mM
  • Used biomass, concentration of ammonia in the feed and residual ammonia concentration to calculate ammonia flux
    • formula used to calculate flux= [dilution rate x (input ammonia concentration