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Project Guides

The Wolbachia Project is organized as a five-part lab series. Each Project Guide has been designed to facilitate inquiry-based research experiences and optimize student success in the classroom.

 

Introductory Activity (Optional)

Wolbachia and Reproductive Parasitism

This activity uses manipulatives to explore the associations between Wolbachia and arthropod hosts. As a result, students will unravel the mystery of this host-microbe symbiosis and investigate its relevance to human health.

 

Lab 1: Arthropod Collection & Identification (Biodiversity)

Goal:
To introduce students to the spectacular diversity and abundance of arthropods and to prepare specimens for DNA analysis.

Learning Objectives:gallery1-7
Upon completion of this activity, students will conduct field work to collect insects from local fauna, appreciate the ubiquity of symbiotic microbes in animals, understand how to use a taxonomic key to identify insects to Order, and prepare lab notes and specimens for molecular studies.

Teaching Time: 60-90 minutes


Student Guide: Arthropod Collection & Identification (6/21) (.docx / .pdf)


 

Lab 2: DNA Extraction (Biotechnology)

Goal:
To introduce students to DNA extraction techniques and to isolate genomic DNA from arthropods and Wolbachia, the endosymbiotic bacteria that live within the cells of about 40% of insect species.

Learning Objectives:
Upon completion of this activity, students will transition from field work and morphological classification (Lab 1) to molecular biology and biotechnology, learn about DNA as a diagnostic tool to discover unseen microbes, increase abilities in biotechnology, and understand the process of inquiry and discovery-based research. They will isolate total genomic DNA from arthropods identified in the Lab 1.

Prerequisite Skills: Prior practice with micropipettors.

Teaching Time: 90 minutes (2 class periods – optional 45min stopping point on page 7)


Qiagen DNeasy Blood & Tissue Kit (recommended)

Edward’s Buffer

Promega Wizard SV genomic DNA purification system


 

Lab 3: Polymerase Chain Reaction (Biotechnology)

Goal:
To screen for Wolbachia symbiont DNA in the extracted DNA from insects using one of the most widely used biotechnology techniques in biological research, the Polymerase Chain Reaction (PCR). PCR amplifies DNA millions of times in just a few hours, so that the DNA becomes easy to detect and study in any fashion.

Learning Objectives:gallery2-5
Upon completion of this activity, students will use and understand one of the most useful biotechnology tools in the life sciences (PCR), understand DNA as the hereditary basis of life, utilize DNA as a diagnostic tool to discover microbes, and seamlessly transition their discovery-based science from organisms to molecules. Students will amplify DNA extracted from experimental and control arthropods using Polymerase Chain Reaction (PCR).

Prerequisite Skills: Prior practice with micropipettors.

Teaching Time: 45 minutes


standard PCR (Recommended)

This lab activity requires two separate PCR reactions (Arthropod and Wolbachia) which allows for customization of the annealing temperature. The protocol is recommended for more sensitive Wolbachia detection.

  • Student Guide: Standard PCR (6/21) (.docx / .pdf)

duplex pcr (alternate protocol)

This lab activity condenses the PCR protocol into one duplex reaction that amplifies both arthropod and Wolbachia DNA in the same assay. It will amplify most, but not all, Wolbachia as the annealing temperature is optimized for the arthropod DNA. The protocol is recommended for efficient use of classroom time and resources.

  • Student Guide: Duplex PCR (6/21) (.docx / .pdf)

pcr calculators (optional quick-tools)

Note regarding the PCR protocol


 

Lab 4: Gel Electrophoresis (Biotechnology)

Goal:
To determine the presence or absence of PCR products and quantify the size (length of the DNA molecule) of the product.

Learning Objectives:gallery4-4
Upon completion of this activity, students will have integrated scientific discovery, inquiry and biotechnology. Students will understand that DNA contains hereditary information in the form of genes, how DNA samples separate-based upon different sizes, and learn how to stain and visualize DNA samples. We will be using agarose gel electrophoresis to determine the presence and size of the Wolbachia 16S rRNA gene amplified by PCR.

Teaching Time: 90 minutes or two class periods of 45min each


MiniOne System

  • Lab Activity: MiniOne Gel electrophoresis (6/21) (.docx / .pdf)

Standard Gel Electrophoresis

  • Lab Activity: Standard Gel Electrophoresis (6/21) (.docx / .pdf)

 

Lab 5: Bioinformatics

Goals:
Bioinformatics I: To analyze and interpret the quality of Sanger sequences, and generate a consensus DNA sequence for bioinformatics analyses.NCBI

Bioinformatics II: (1) To show the ways in which the NCBI online database classifies and nucleutide-blast-cover copyorganizes information on DNA sequences, evolutionary relationships, and scientific publications. (2) To identify an unknown nucleotide sequence from an insect endosymbiont using the NCBI search tool BLAST.

Teaching Time: 90 minutes (one class period for each activity)


I. Sanger Sequence Analysis

II. NCBI Taxonomy & BLAST Searching

Required resources: DNA Sequence Files