Elizabeth Polidan Week3: Difference between revisions
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**Northern analysis | **Northern analysis | ||
***Measured RNA to determine if levels varied with ammonia concentration in nitrogen regulated genes | ***Measured RNA to determine if levels varied with ammonia concentration in nitrogen regulated genes | ||
**** | ****GDH1, GDH2 | ||
**** | ****GAP1, PUT4 | ||
**** | ****ILV5, HIS4, GLN1 | ||
**Enzyme activities | **Enzyme activities | ||
*** | ***Levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were measured to determine if there was any change in enzyme activity as the ammonia concentration was varied. | ||
*Discussion | |||
**What do the X and Y axes represent? | **What do the X and Y axes represent? | ||
**How were the measurements made? | **How were the measurements made? | ||
**What trends are shown by the plots and what conclusions can you draw from the data? | **What trends are shown by the plots and what conclusions can you draw from the data? | ||
* | *Conclusion | ||
**It is the ammonia concentration that regulates nitrogen metabolism in S. cerevisiae, not the flux level. | |||
***The driver of the observed changes was either ammonia concentration itself (intra- and intercellular) or ammonia concentration-driven changes in levels of metabolites such as glutamate or glutamine. |
Revision as of 21:07, 30 January 2013
Elizabeth Polidan
BIOL 398.03 / MATH 388
- Loyola Marymount University
- Los Angeles, CA, USA
Vocabulary
- Permease
- (Science: enzyme) general term for a membrane protein that increases the permeability of the plasma membrane to a particular molecule, by a process not requiring metabolic energy. Reference: http://www.biology-online.org/dictionary/Permease
- Allosteric inhibition
- See allosteric Enzyme. Inhibitors act as 'modulators' in enzyme execution as they can attach themselves to a molecule that will alter the binding Site for the enzyme, rendering it unusable and therefore rendering the enzyme inactive. Reference: http://www.biology-online.org/dictionary/Allosteric_Inhibitors
- Northern analysis
- A procedure similar to the southern blot analysis, used mostly to separate and identify rNA fragments; typically via transferring RNA fragments from an agarose gel to a nitrocellulose filter followed by detection with a suitable probe. Reference: http://www.biology-online.org/dictionary/Northern_blot_analysis
Article Outline
- What is the main result presented in this paper?
- What is the importance or significance of this work?
- What were the limitations in previous studies that led them to perform this work?
- Methods
- Saccharomyces cerevisiae was grown in cultures with continuous feed
- Incoming concentrations of ammonia were varied from a low of 29 milimoles per liter (mM) to a high of 118 mM.
- Incoming concentrations of glucose were all held at 100 mM
- The dilution rate was 0.15/hour
- Physiological parameters were monitor to determine the state of the yeast in the system
- Biomass was measured in grams/liter
- Residual ammonia was measured mM
- Ammonia flux was calculated from resulting biomass, input ammonia concentration and residual ammonia concentration: NH4 flux = (diluation rate x (incoming ammonia concentration – residual ammonia concentration)/biomass)
- Oxygen consumption was measured in mM per gram biomass per hour (mM/g/h)
- Carbon Dioxide production was measured in mM/g/h
- Respiratory quotient was calculated as (CO2 produced/O2 consumed)
- Measured alpha-ketoglutarate at varying ammonia concentration
- Measured glutamate at varying ammonia concentration
- Measured glutamine at varying ammonia concentration
- Northern analysis
- Measured RNA to determine if levels varied with ammonia concentration in nitrogen regulated genes
- GDH1, GDH2
- GAP1, PUT4
- ILV5, HIS4, GLN1
- Measured RNA to determine if levels varied with ammonia concentration in nitrogen regulated genes
- Enzyme activities
- Levels of NADPH-glutamate dehydrogenase, NAD-GDH, and GS activity were measured to determine if there was any change in enzyme activity as the ammonia concentration was varied.
- Saccharomyces cerevisiae was grown in cultures with continuous feed
- Discussion
- What do the X and Y axes represent?
- How were the measurements made?
- What trends are shown by the plots and what conclusions can you draw from the data?
- Conclusion
- It is the ammonia concentration that regulates nitrogen metabolism in S. cerevisiae, not the flux level.
- The driver of the observed changes was either ammonia concentration itself (intra- and intercellular) or ammonia concentration-driven changes in levels of metabolites such as glutamate or glutamine.
- It is the ammonia concentration that regulates nitrogen metabolism in S. cerevisiae, not the flux level.