Msaeedi23 Week 11

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Process for GenMapp analysis

Microarray paper: <http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115243> Genome Paper: <http://www.nature.com/ng/journal/v35/n1/full/ng1227.html>

Week 11

  • For the microarray paper (GenMAPP Users only), include the following:
  • Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.
  • Determine the sample and data relationships, i.e., which files in the data correspond to which samples in the experimental design.
  • Construct a flow chart that illustrates the above.

Unknown Term Definitions

  • Phosphorelay: A multi-stage process, involving the movement of phophoryl groups by histidine kinases in bacterial signal transduction.

< https://en.wiktionary.org/wiki/phosphorelay>

  • Putative: Believed or deemed to be the case; accepted by supposition rather than from proof.

< https://en.wiktionary.org/wiki/putative>

  • Supernatant: Floating on the surface or over something; the chemistry of a liquid-lying above a sediment or settled precipitate.

< http://dictionary.reference.com/browse/supernatant?s=t>

  • Chemiluminescent: The emission of light as the result of a chemical reaction.

< https://en.wikipedia.org/wiki/Chemiluminescence>

  • Endogenous: Growing or developing from within; originating within.

< http://dictionary.reference.com/browse/endogenous>

  • Non-Permeabilized: Made not permeable, unable to infiltrate a surface.

< https://en.wiktionary.org/wiki/nonpermeabilized>

  • Albeit: Although, even if.

< http://dictionary.reference.com/browse/albeit?s=ts>

  • Attenuated: To render less virulent, as a strain of pathogenic virus or bacterium.

< http://dictionary.reference.com/browse/attenuated?s=t>

  • Modulating: To regulate or adjust to a certain measure or proportion; soften or tone down.

< http://dictionary.reference.com/browse/modulating?s=t>

  • Homodimerization: Any reaction which leads to the formation of a homodimer, a molecule composed of paired identical proteins.

< http://dictionary.reference.com/browse/modulating?s=t>

Article Outline

Write an outline of the article. The length should be a minimum of the equivalent of 2 pages of standard 8 1/2 by 11 inch paper (you can use the "Print Preview" option in your browser to see the length). Your outline can be in any form you choose, but you should utilize the wiki syntax of headers and either numbered or bulleted lists to create it. The text of the outline does not have to be complete sentences, but it should answer the questions listed below and have enough information so that others can follow it. However, your outline should be in YOUR OWN WORDS, not copied straight from the article.

    • What is the importance or significance of this work (i.e., your species)?
    • What were the methods used in the study?
    • Briefly state the result shown in each of the figures and tables.
  • Table 1: Lists all of the B. Pertussis strains used in the study along with their genotypes/relevant features, and the source where the strain was obtained.
  • Table 2: A complete list of all of the forward and reverse primers used in cloning are provided with their sequences, 5'-3', and their indicated mode of amplification (forward or reverse).
  • Table 3: Lists specific reverse and forward primers used in the real-time PCR analysis.
  • Figure 1: Two biological replicates were performed and the expression of the capsule locus was monitored using real-time PCR and observed to be significantly up-regulated in the mouse lungs compared to its in vitro Bvg+ expression level, with a peak of expression at day 3 p.i. A similar trend of expression was seen for another bvg-repressed gene, vrg6. Concurrently, the expression of vags including bvgA, fhaB and ptx were all up regulated at day 3 and day 7 p.i. compared to Day 0 and to the in vitro Bvg+ expression level.
  • Figure 2: Outlines the construction of the kpsT, kpsE, vipC, and BPSM B. pertussis mutants. Goes on to portray the Southern blot analysis and the transcription efficacy of the downstream gene. Finally it looked at the growth kinetic profiles of each mutant. "The ΔkpsT, ΔkpsE and ΔvipC mutant strains displayed hemolytic and domed colony morphology on blood agar plates (data not shown) and in vitro growth kinetics comparable to the parental BPSM strain (Fig. 2 D), implying that these single gene deletions within the capsule locus did not affect the in vitro fitness of the bacteria." <http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115243#pone-0115243-g002>
  • Figure 3: Depicts phenotypic characterization of the kpsT, kpsE, and vipC mutants. It was observed that ΔkpsT, ΔkpsE and ΔvipC cells consistently displayed levels of fluorescence shift comparable to that measured with KOcaps, corresponding to nonspecific background shift. "The FACS data supports that in-frame deletion of the single ORF kpsT, kpsE or vipC within the capsule locus is sufficient to prevent the presence of the PS capsule at the bacterial surface."
  • Figure 4:Compared to BPSM, the band signal intensity for FHA in ΔkpsT 10x concentrated culture supernatant was about 30% down-regulated (Fig. 4 A). In contrast, signal intensities for FHA in the whole cell lysates appeared comparable between mutant strains (Fig. 4A), indicating that secretion into the culture supernatant may be compromised in ΔkpsT. Similarly, secretion but not production of PT appeared to be impaired in ΔkpsT (Fig. 4C). "In contrast, both production and secretion of BrkA in ΔkpsT were visibly reduced (Fig. 4 B). Production/secretion levels of FHA, PT and BrkA were partially restored to parental level in ΔkpsTcom strain (Fig. 4 A, B and C)."
  • Figure 5: Consistent with the Real-time PCR analysis, expression of the bvgAS locus and fhaB was not found to be down-regulated in the ΔkpsT mutant (S1 Table).In addition to further support this analysis, the microarray data revealed that the absence of KpsT affects negatively the expression of a large number of bvg-regulated genes. "Such overall down-regulation is likely to be responsible for the attenuated phenotype observed with the ΔkpsT mutant in mice."
  • Figure 6: "Higher amounts of BrkA, PT and to a lesser extent FHA were detected with the BvgS-VFT2- ΔkpsT double mutant compared to ΔkpsT single mutant, with band signal intensities comparable to those observed for wild type BPSM and BvgS-VFT2 strain (Fig. 6A). Also, real-time PCR analysis showed that down-regulation of the brkA, ptx and sphB1 genes observed with the ΔkpsT single mutant was not seen with the BvgS-VFT2- ΔkpsT double mutant (Fig. 6B). These data thus indicated that deletion of kpsT in a Bvg+-phase locked mutant did not affect the virulence genes expression therefore suggesting that VFT2 mutation in the BvgS sensor is dominant over kpsT deletion. Consistently, a parental in vivo virulent phenotype was observed with BvgS-VFT2- ΔkpsT double mutant in mice (Fig. 6C). Altogether, these results indicate that deletion of kpsT ORF in a constitutive bvgS background had no effect on the expression of bvg-regulated genes."
  • Figure 7: "Western blot analysis using anti-His and anti-BvgS antibodies confirmed the identity of the 140 kDa eluted protein as His-BvgS monomers in E2 and E3 from BPSH but not from BPSM extracts (Fig. 7B). A band at 50 kDa MW was also observed in E3 and E4 from both BPSM and BPSH, suggesting that this unknown protein bound to the Ni-NTA beads likely due to high His content (Fig. 7A). A stronger signal intensity of the 140 kDa band was observed under reducing/heat treatment conditions (Fig. 7C). Altogether these data supported that KpsT is necessary for BvgS oligomerization although no direct physical interaction seems to occur between both proteins."
  • Figure 8: The results portrayed in this figure support that neither KpsT alone nor the KpsMT complex is enough to restore fully a parental colonization profile in KOcaps. "Therefore, instead of KpsT alone, the entire capsule transport machinery may play a role in the BvgS-mediated signal transduction and indirectly in the overall bacterial virulence."
    • How do the results of this study compare to the results of previous studies (See Discussion).
  • For the microarray paper (GenMAPP Users only), include the following:
    • Describe the experimental design of the microarray data, including treatments, number of replicates (biological and/or technical), dye swaps.
      • This article linked a recent resurgence of pertussis with an expansion of B. pertussis strains containing a novel allele for the pertussis toxin (ptx) promoter ptxP3 in place of the typical ptxP1 promoter. PtxP3 strains were noted to have better fitness due to higher expression rates of the pertussis toxin. In this experiment, a microarray analysis was conducting comparing several ptxP1 strains to several ptxP3 strains.
  • Treatment Group: PtxP3 strains (n=5)
  • Control Group: PtxP1 strains (n=9)
  • Three or more biological replicates were conducted for each of the 14 "Bordetella pertussis" strains assessed in this paper, making for a total of 57 unique biological replicates. The data collected for all the replicates within each group was averaged and compared for the final microarray analysis.
  • Replicates, including strain names and the number of biological replicates per strain in parentheses:
      • 1949-B0558 (5)
      • 1967-B1213 (3)
      • 1982-B0689 (4)
      • 1988-B2973 (5)
      • 1995-B0602 (4)
      • 1995-B0607 (4)
      • 1996-B0777 (4)
      • 1999-B1834 (4)
      • 2000-B1878 (4)
      • 2000-B1917 (3)
      • 2007-B3104 (4)
      • 2008-B3183 (5)
      • 2008-B3234 (4)
      • 2008-B3265 (4)
  • Determine the sample and data relationships, i.e., which files in the data correspond to which samples in the experimental design.
  • Construct a flow chart that illustrates the above.


DOCUMENT ALL STEPS TAKEN, MANIPULATIONS OF FILES AND ALL.

1. Experimental design-what is the treatment and what is the control

  • Biological vs technical replicants- for vibrio control was the untreated strain, technical replicates = splitting a sample, different from biological replicates. Biological replicates are derived from the original strain. *note how many replicates of each. # of chips(microarrays) (for vibrio it was 9, total number of samples)
      • Dye swaps, cy3 green dye and cy5 red. note how many swaps. Swaps done to avoid dye playing an effect in replication.
        • Draw a diagram to help of how chips were obtained.

2. Sample + data relationship. In raw data there should be one file to each chip. Match file name to sample. raw.zip on arrayexpress has one file for each chip. Sdrf.txt open in excel to show whats in the samples-should tell you all of the samples, their matches, and dye swaps.

3. Compiled raw data file. Include columns for: ID, Sample log2FC, sample log2FC. Compiling raw data files into one spreadsheet to prepare for statistical analysis.

4. Normalization + statistical analysis. Customized for each dataset due to different treatments. Either t-test or one way anova. Also due sanity check, which will be a result in the paper. Compare it to what was written in the paper, corresponding to specific genes. Comparing log-fold changes of specific genes between our data and the paper's.

5. GenMAPP + mappfinder. Run data through database created by groupmates.

6. Deliverables-describing methods, production of tables including the sanity check, etc.

  • In discussion compare what we found vs to what they found.

Mahrad Saeedi

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