Natalie Williams: Electronic Lab Notebook

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Revision as of 23:13, 25 January 2015 by Natalie Williams (talk | contribs) (Addition of C and Northern Analysis)
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Electronic Lab Notebook

This page is dedicated to the course I am taking at Loyola Marymount University this Spring semester. The course is Biomathematical Modeling. Each week there will be an assignment that I will post as well as a link to a Class Journal Entry.

Week 1

So that my peers and professors can learn more about myself, I am answering a few questions about my interest in Biology and Mathematics.

  1. My Favorite Aspect of Biology
    • I enjoy biology for many reasons. The main reason is because it tells the story of life. Through biology, we have learned not only about ourselves as humans, but we have also gained knowledge about other unicellular and multicellular organisms. This information helps guide how we live today as well as what we can predict for the future. Micro- to macroscopic, biology connects the dots and we continue to discover amazing things about our world.
  2. My Favorite Aspect of Mathematics
    • Math just makes sense to me. It is very logical and systematically explains various processes and theories. Numbers do not lie. They provide evidence to why we can believe what we believe. Math further supports and strengthens conclusions made in the sciences like biology.


Click the following to read my response for the Week 1's Class Journal Entry

Week 2

Biological Terms

These are the following terms that I had a hard time fully understanding. I did not know exactly what they meant or what they were in relation to what was done in the article.

  • Proline: a heterocyclic, non-polar amino acid that is present in all proteins studied to date [Source]
  • Continuous Cultures: a technique for production of microbes or microbial products in which nutrients are continuously supplied to the fermenter [Source]
  • Assimilation: The conversion of nutriment into a useable form (e.g. liquid or solid) that is incorporated into the tissues and organs following the processes of digestion [Source, (3)]
  • Permease: protein in bacteria that is responsible for transport of substances into or out of the cell; sometimes referred to as a transport protein [Source]
  • Northern analyses (RNA): also known as Northern blot analysis; a procedure used mostly to separate and identify RNA fragments; typically the RNA fragment is moved to an agarose gel to a nitrocellulose filter [Source]
  • Oligonucleotides: a short polymer of nucleotides [Source]
  • GS (Glutamate synthesase) transferase: An enzyme that catalyses a reaction between ATP, l‐glutamate, and NH3 to form ADP, orthophosphate, and l‐glutamine [Source]

Outline

Introduction

  • Yeast uses ammonia more than other sources of nitrogen
  • Breakdown of nitrogen is seen on the level of gene expression and enzyme activity
    • The studies have show that the fluctuation of nitrogen influences various cultures rather than the concentration of nitrogen (ammonia)
  • In this study, the yeast all intake and remove ammonia at the same rates but the amount that each culture is exposed to varies by concentration

Physiological parameters

  • The same culture of yeast is used by under these concentrations:
    • A fixed glucose concentration
    • Dilution rate of .15 h-1
  • An increase of ammonia saw an increase in the biomass of the culture
  • However, there was nitrogen limitation in that after increasing beyond 61 mM, the biomass remained the same (8.2 L)
    • As a result, there was leftover ammonia/feed
    • Glucose become limited resource
  • Ammonia going into biomass occurred generally at 1.1 milli moles g-1
  • The levels of CO2 and O2 being produced and consumed respectively saw minimal changes and were nearly constant
  • No significant changes were seen in the breakdown of carbon when the ammonia concentration switched from limited to excess
  • As the ammonia concentration increases the cells increases the production and concentration of glutamine and the glutamate

Northern Analysis

  • These tests were used to see if the increase in nitrogen had any effect on genes
  • The responses of genes that intake ammonia were observed and recorded to see how they would react to the varying concentrations
  • For specific genes, the gene was either repressed (GDH1) or stimulated (GDH2)

Enzyme Activity

  • NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were recorded to see how they reacted to the changes in ammonia concentrations
  • When ammonia concentrations increased from 29 to 118 mM, the activity of NADPH-GDH decreased
    • Following this decline, the production of GDH1 fell as well
  • There was a sharp increase in the level of NAD-GDH between 29 and 61 mM, but as the concentrations increased, no change was seen
    • Both of these results suggested that the RNA expression of the affected proteins as well as the electron carrier transporters were controlled at a transcriptional level
  • GS transferase and GS activity has an inverted relationship with the concentration of ammonia up to 61 mM
  • However, GS activity did not seem to be affected by transcription, so it must have posttranscriptional alterations.
  • NADPH and GS activity affect ammonia, α-ketoglutarate, glutamate, and glutamine concentrations

Conclusions

  • This data shows that ammonia concentration affects nitrogen metabolism on the transcriptional and enzymatic levels in yeast
  • Nitrogen metabolism can be affected either by intracellular or extracellular ammonia concentrations or by altering glutamate, glutamite, and α-ketoglutarate levels
  • Don’t seem to know what really affects nitrogen metabolism but what it affects
  • Suggests that if it is the first (controlled by ammonia concentrations), then yeast would have to have an ammonia sensor that helps it react to the nitrogen concentrations in its environment

Figures

A

  • X: NH4 concentration (mM)
  • Y axes: Residual NH4 concentration (mM)
    • Biomass (g-1)
    • NH4 flux
  • Can see that the NH4 flux was nearly constant throughout the changing of NH4 concentrations; 1mmol/(gxh)
  • At 60 mM there was no ammonia left, but as the concentration increased, the residual NH4 increased almost linearly
  • There was a sharp increase, 4 to 8, in the biomass from 20 – 60 mM of NH4 and the increase of the NH4 after 60mM just caused a fluctuation between 7 and 9.

From this figure, we can see that increasing the ammonia concentration impacts the growth rate initially, but after a certain concentration, no significant change can be seen.
B

  • X: NH4 concentration (mM)
  • Y axes: O2 consumption (mmol/gh)
    • CO2 production (mmol/gh)
    • Respiratory Quotient
  • When there are low concentrations of ammonia, O2 is consumed until there is at least 40mM of NH4.
  • However, for CO2, its production decreases at low concentrations of ammonia. When it reaches ~60mM of NH4, the levels out and fluctuates around 4.5.
  • The respiratory quotient shows change when both the O2 and CO2 levels change; yet, when O2 begins to level off, the quotient stabilizes.


C Each of the graphs shows the level of their respective amino acids: Glutamate, glutamite, and α-ketoglutarate

  • X: NH4 concentration (mM) for each of the graphs
  • Y: α-ketoglutarate (micromol/g)
    • Glutamate (micromol/g)
    • Glutamine (micromol/g)
  • For α-ketoglutarate, the graph shows that its levels decrease as the concentration of NH4 increases until it fluctuates at 60 mM.
  • Glutamate seems to be constant until ~45 mM NH4, where it jumps and continues to increase as the ammonia concentration grows.
  • The glutamine appears to climb linearly as the concentration of ammonia also increases.

One can conclude that the general trend is that there is a positive relationship among these amino acids while α-ketoglutarate has an inverted relationship. Plus, ammonia concentrations affect intracellular metabolisms.
Northern Analysis

  • X: NH4 concentration (mM)
  • Y: Percent expression
  • The left panel had GDH1 and GDH2 depicted
    • GDH1 had a general decline as the concentration of ammonia increased.
    • GDH2 had a varied data set, but the trend seemed to be an increased when compared to initial concentration.
  • The central panel had GAP1 and PUT4. Both seemed to have started with the same level of expression
    • GAP1 saw a slight increase in expression, but after ~45 mM concentration of NH4, its expression level fell.
    • PUT4’s expression increased sharply until the NH4 concentration reached ~45 mM, and then proceeded to plummet. However, its expression remained above GAP1’s.
  • The right paneled shows the expression of GLN1, HIS4, and ILV5.
    • GLN1’s expression rose linearly until it reached its peak at 60 mM before declining.
    • HIS4 had varied expression levels with various peaks. The highest level of its expression came when the ammonia concentration was around 80 mM.
    • The expression of ILV5 increased slowly before falling at an ammonia concentration around 60 mM.

The gene expression of each of these genes seemed to lower overall, except GDH2 and GLN1 whose expressions increased compared to their starting expressions.


Back to User Page: User:Natalie Williams
To view the Course and Assignments:BIOL398-04/S15


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