Educational Level:

Roadmap Objectives:

• • Article: Novembre J, Pritchard JK, Coop G. (2007) Adaptive drool in the gene pool. Nat Genet. Oct;39(10):1188-90.
  • Content area/major concepts: This paper is a news and views article in Nature Genetics that provides perspective for the remaining papers in the module. It will introduce some central concepts and provide a way for students to become familiar with some C.R.E.A.T.E. tools.
    
    amylase, copy number variation (CNV), gene flow, genetic variation, haplotype variation (skip?), population genetics, selective pressure, single nucleotide polymorphisms (SNPs)
  • Methods or technology used to obtain data:
  • How the CREATE strategy was used:
  • Biggest teaching challenge: This article uses a number of population genetics concepts. Most but not all of the class will have had genetics, but even those with genetics will not be acquainted with the population genetics issues in this article
• • Article: Perry GH, Dominy NJ, Claw KG, Lee AS, Fiegler H, Redon R, Werner J, Villanea FA, Mountain JL, Misra R, Carter NP, Lee C, Stone AC. (2007) Diet and the evolution of human amylase gene copy number variation.Nat Genet. Oct;39(10):1256-60.
  • Content area/major concepts: This paper was chosen as the next one in the module to introduce one possible reason for variation in amylase activity. By the time students use this paper they will have conducted amylase assays using saliva samples from themselves and their lab partner. The information in this paper will provide background they can use when writing up a lab report using this data.
    
    Copy number variation, correlations, using PCR for quantitative as opposed to qualitative measurements, genetic drift, population log2 ratios, null hypothesis, fitness
  • Methods or technology used to obtain data: Quantitative PCR, electrophoresis, Western blotting, fiber FISH (fluorescent in situ hybridization), Cot values (?), aCGH (comparative genome hybridization) analysis
  • How the CREATE strategy was used:
  • Biggest teaching challenge: All or most of the class will be familiar with analysis of band size in end-point PCR. For most of the class, quantitative PCR will be a new concept, and this technique is not explained in the paper. However, 3 of 24 students enrolled in the class for Fall 2012 have used quantitative PCR. I would be surprised if any class members have heard of omparative genome hybridization.
• • Article: Mandel AL, Peyrot des Gachons C, Plank KL, Alarcon S, Breslin PA. (2010) Individual differences in AMY1 gene copy number, salivary α-amylase levels, and the perception of oral starch. PLoS One. Oct 13;5(10):e13352.
  • Content area/major concepts: The introduction for this paper discussed possible physiological reasons for variations in α-amylase activity, reminding students that changes in protein expression are not necessarily due to genetic changes, an opportunity for a broader discussion of this issue. The paper then measures specific activity of α-amylase in different individuals using a different enzyme assay than that used by students in lab. The genetic variation is then linked to perceptual differences when a viscous amylase solution is digested in the mouth.
    
    specific activity of enzymes, copy number variation, quantitative PCR, subjective vs. objective measurements, qualitative vs. quantitative assays, viscosity, measures of sample variation
  • Methods or technology used to obtain data: rheological measures of viscosity, Western blotting, enzyme assays
  • How the CREATE strategy was used:
  • Biggest teaching challenge: Students are likely to find the biophysical measurements of viscosity confusing. However, it is not necessary for them to understand the theory behind these measurements to understand the key ideas in the paper.
• • Article: Dang S, Hong T, Bu D, Tang J, Fan J, Zhang W. (2012) Optimized refolding and characterization of active C-terminal ADAMTS-18 fragment from inclusion bodies of Escherichia coli. Protein Expr Purif. Mar;82(1):32-6. Background Reading for Paper 4: Burgess RR. (2009) Refolding solubilized inclusion body proteins. Methods Enzymol. 2009;463:259-82.
  • Content area/major concepts: Although this paper is not directly related to the previous three papers, it will help the students with the laboratory exercises related to these papers. One of the goals in the laboratory is for the students to make a GST/amylase fusion protein. Students were successful in making fusion clones in 2011, and E. coli with the fusion clone showed increase amylase activity. However, cell lysates did not show any evidence of amylase. Many GST fusion proteins aggregate and form insoluble inclusion bodies, so students will test the hypothesis that this is what occurs with amylase-GST fusions. If these experiments indicate that inclusion bodies have formed, the papers above will help the students develop ways to recover the fusion proteins in soluble form.
    
    Expression vectors, fusion proteins, lac operon regulation, inducible genes, artificial inducers; affinity chromatography
  • Methods or technology used to obtain data: Recombinant clone construction, bacterial transformation, sonication, protein denaturation, protein folding, glutathione-Sepharose chromatography, electrophoresis, Western blotting, mass spectroscopy, platelet aggregation assay
  • How the CREATE strategy was used:
  • Biggest teaching challenge: Understanding why the different conditions were selected for protein refolding, extrapolating from a different fusion protein to the GST-amylase protein construct made by the students.

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