Emilysimso Week 11

From LMU BioDB 2015
Jump to: navigation, search

Experimental Design

  • Control: wild-type S. oneidensis MR-1
  • Treatment: used 2,2'-dipyridyl diluted to 80, 160, 240, and 320 micro-molar concentrations
    • This is to reduce the iron uptake abilities of the samples
  • Biological treatments - all of the studies were done in triplicates
    • Three samples per treatment
  • Number of chips: 12 (3 samples x 4 treatments = 12)
  • Dye swaps - unable to find evidence of this in the paper

Vocab Definitions

  1. Putative: entity or concept that is based on what is generally accepted or inferred even without direct proof
  2. Alcaligin: dihydroxamate siderophore, involved with important pathogens
  3. Enzyme prosthetic group: required for enzyme function
  4. Iron chelating: detoxify metal ions and prevent poisoning, removing excess iron
  5. Siderophores: ferric ion specific chelating agent, make iron available to cell
  6. Heme: nonprotein, insoluble, iron protoporphyrin constituent of hemoglobin; iron compound, pigment portion or protein-free part of hemoglobin
  7. Orthology: homologous genes related by speciation
  8. 2-2'-dipyridyl: chelator of iron
  9. Lag phase: the delay before bacteria start exponential growth
  10. Mid-log phase: the maximum rate of reproduction, number of bacteria increases directly with time

Outline

  • What is the importance or significance of this work (i.e., your species)?
    • Studying the iron homeostasis of Shewanella oneidensis is relevant because this bacteria is a pathogen to both fish and humans (Yang et al., 2). Other species, such as Vibrio cholerae, which is also a pathogen, were previously studied and researchers found that “iron-regulated metabolisms are essential” for their virulence (2). Therefore, looking at the iron uptake of S. oneidensis could help researchers understand how this pathogen interacts with humans.
  • What were the methods used in the study?
    • First, the researchers amplified a strand of wild-type S. oneidensis MR-1 using PCR. They confirmed that they had the desired sequence using DNA sequencing. (14)
    • To test the uptake of iron in aerobic versus anaerobic conditions, the MR-1 was first grown to mid-log phase and diluted. They then created diluted solutions of 2,2’-dipyridyl in 80 micro-M, 160 micro-M, 240 micro-M, and 320 micro-M concentrations. This was done in triplicates for each condition. Next, ferrous sulfate solution was added to replete the iron. They used a Luria-Bertani medium at 30 C for the aerobic culturing and supplemented this medium with 10mM lactate and an electron acceptor for the anaerobic culturing. (14)
    • To test the iron reduction rate, cells were grown anaerobically to mid-log phase in the LB medium along with 10mM fumarate and 10mM lactate. They were transferred to a solution of 5mL LB medium, 10mM lactate, and 10mM Fe(III) dioxide. They then performed the ferrozine assay. (14).
    • For the microarray portion of the experiment, four biological replicates were grown in the LB medium. 2,2’-dipyridyl was added at concentration of 160 micro-M, followed by ferrous sulfate after 60 minutes. They then centrifuged to allow for RNA extraction.
  • Briefly state the result shown in each of the figures and tables.
    • Figure 1: Graph A shows the results of the iron-chelating portion of the experiment. The bacteria was able to grown, with lessened ability, through the 240 micro-M concentration of the 2,2’-dipyridyl. However, it was unable to grow in the 320 micro-M condition. Graph B shows the results of adding the 2,2’-dipyridyl to the bacteria but then reversing the results with FeSO4, which is used to replete iron concentrations. The strains were able to grow in each of the shown conditions. (3)
    • Table 1: This table shows the results for each of the studied genes in both the microarray and qPCR assays. Statistical tests were done to see the correlation between these studies. (4)
    • Table 2: This table shows the amount of up-regulated and down-regulated genes at each time point for the various conditions of either depletion or repletion. (5)
    • Figure 2: These two graphs show the results of the iron depletion state (A) and the repletion state (B) grouped by functional category of the genes. (6)
    • Table 3: This table shows the results of the studied genes in iron acquisition, anaerobic energy metabolism, and protein degradation categories based upon how they changed their expression in the iron depletion and repletion conditions. This table also gives descriptions of what each gene is responsible for in the bacteria. (7-8)
    • Figure 3: This figure shows the relationships between the various genes and how they acquire iron. Some of the genes are known, while others are unknown. (9)
    • Table 4: This table shows how certain genes are related to protein synthesis and aerobic energy metabolism in the iron depletion and repletion conditions. The given values show how strongly the genes correlate to the given pathways. (10)
    • Figure 4: The researchers looked at SO3032 to test the importance of iron acquisition genes under iron depleted conditions (DSP10 is parent strand). SO3032 was unable to grow in the presence of 120 micro-M iron chelator (A) and had only some growth under 160 micro-M conditions - less than those shown in graph A (B). Thus, a cell needs SO3032 for survival of iron starvation.Graph C shows a gene encoding a protein - SO2017 (which is located in the same operon as the heat shock protein). The mutant strand cannot grow in the presence of 2,2’-dipyridyl, therefore, a lack of SO2017 prohibited the cell’s ability to survive iron depletion.
    • Figure 5: Figure A looked at the disruption of SO1415 impared anaerobic Fe(III) reduction - produced less than half the amount of Fe(II). Figures B and C saw that SO1415 is involved in regulating specific branches of anaerobic energy metabolism - used 10 mM lactate as the electron donor and a non-metal electron acceptor (5 options). Both show significant growth deficiency.
  • How do the results of this study compare to the results of previous studies (See Discussion).
    • “Anaerobic energy metabolism module for iron experiments conducted under aerobic conditions” (12) – ‘‘S. oneidensis’’ is able to still grow under iron depleted conditions. Additionally, a number of genes involved in the aerobic response where induced by iron depletion, while some genes involved in anaerobic response were not involved in iron depletion (12).
      • This is different from other organisms and may be because anaerobic energy metabolism may be an iron-storage mechanism to release previously stored iron.
      • However, this has been found in ‘‘E. coli’’
    • Crp in ‘‘E. coli’’ regulated biological processes and responds to glucose levels but in ‘‘S. oneidensis,’’ Crp is a part of anaerobic energy metabolism. (12)
    • ‘‘S. oneidensis’’ did not go through oxidative stress as part of the study, which was expected. (12)
    • SdhA and AcnA (which are controlled by Fur and RNA RyhB during iron uptake) were not observed in ‘‘S. oneidensis’’ as expected (12)
    • Overall, there are more transcriptional factors that Fur needed for iron response (13)
  • Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.
    • Treated MR-1 cultures with 160 micro-M 2,2’-dipyridyl then, after 1 hour, added ferrous sulfate
    • 4 biological replicates
  • Determine the sample and data relationships, i.e., which files in the data correspond to which samples in the experimental design.
    • Investigation description: describes the methods used for the microarray portion of the experiment
    • Sample and data relationship: this is the file we will use; shows what the samples were treated with, how they responded at various times, the correlated file for the microarray data, the control information, and various other aspects of the experiment
    • Raw data: the data before analysis; the IDs in this data set correspond to the sample and data relationship file
    • Processed data: the data after analysis
    • Array design: gives the values for the array
    • All of these links can be found [here]

Flow Chart

Weekly Assignment Information

User: Emilysimso

Assignments

Individual Journal Entries

Class Journal Entries

Group Project

Heavy Metal HaterZ

Retrieved from "https://xmlpipedb.cs.lmu.edu/biodb/fall2015/index.php?title=Emilysimso_Week_11&oldid=6219"