A laboratory unit investigating 2,4-dichlorophenoxyacetic acid degradation in soils

Teresa Johnson, College of WoosterAmy Treonis, University of Richmond

Diuto Esiobu, Florida Atlantic University

Sample 2,4-Ddegradation pathway

Who degrades 2,4-D in soils?

Pseudomonas spp.Arthrobacter spp.

Objectives:

Audience: Biology Majors in a Microbiology course (intermediate to

upper level)

Students who complete these activities will:

•Explore environmental problems using bioinformatics tools.

•Appreciate metabolic flexibility of microbes through study of their ability to use novel chemicals (xenobiotics) as energy sources.

•Integrate concepts in functional genomics, phylogenetics, and ecology.

Step 1: Finding 2,4-D degraders

•Challenge students to develop an appropriate enrichment culture technique & isolate a 2,4-D degrader in pure culture.

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Step 2: Identification of isolate

•16S rDNA sequencing from pure culture

•BLAST search to identify microbe

Step 3: Functional genomics

•BLAST search for tfdA amino acid sequences (Pseudomonas)

•Use aa sequence from BLAST to perform a JGI searchfor sequenced bacteria that contain the gene to determine whether our isolate has been identified as having tfdA genes.

•Design primers to attempt to amplify tfdA genes from isolatefor sequencing.

•If tfdA genes aren’t present, propose alternative mechanismfor degradation.

•If tfdA genes are present, compare aa or nucleotide sequence similarity between known degraders and our isolate.