This project was prepared as part of a BioQUEST faculty development workshop entitled ASM/BioQUEST Bioinformatics Institute at American Society for Microbiology in March 2007. The BioQUEST Curriculum Consortium is committed to the reform of undergraduate biology instruction through an emphasis on engaging students in realistic scientific practices. This approach is sometimes characterized as an inquiry driven approach and is captured in BioQUEST's three P's (problem-posing, problem-solving, and peer-persuasion). As part of this workshop groups of faculty were encouraged to initiate innovative curricular projects. We are sharing these works in progress in the hope that they will stimulate further exploration, collaboration and development. Please see the following links for additional information:

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"A Microbiological and Bioinformatics Safari: Searching for Cellulolytic Prokaryotes from the Environment and Characterization of their Cellulase Genes."
 
 
Authors          Audiences          Overview           Materials          Resources           Future Directions
 

 


Authors


Anne Bernhard
Connecticut College


Mark Martin
University of Puget Sound


Shannon Hinsa-Leasure
Michigan State University

 
   
 


Possible Audiences:

Mid-level undergraduates (sophomore level) to senior level students. There are also modifications that could allow this project to become appropriate for freshman and nonmajors. Additionally, other institutions participating in this project will have access to "in common" data for remote in silico analysis.

This project introduces several concepts of importance, including: enrichment culture, microbial ecology, transposon mutagenesis, screening for mutants, cloning and sequencing, bioinformatic analysis, enzymology and biochemistry, biotechnology, and energy policy. Because we propose that several investigators at different institutions lead their students on this "safari," a further concept of increasing importance is introduced: collaboration with other researchers.  

 
 


Brief Overview:

Cellulases are enzymes that digest the common polysaccharide cellulose using one of several enzymatic strategies (EC 3.2.1.4). A variety of organisms, ranging from marine to terrestrial, aerobic to anaerobic, possess enzymes with this activity. Cellulases may prove to be of significant economic importance to the process of converting cellulose to ethanol, in other words, the burgeoning biofuel industry.

We propose a multi-institutional effort to identify cellulolytic organisms by enrichment, create transposon mutants lacking cellulase activity (via screening of mutants), obtain DNA sequence from these mutants, and to compare the recovered cellulase-specific gene sequences to known cellulase genes. The 16S rRNA sequence from each cellulolytic isolate will also be obtained, and the phylogenetics of the organism and the cellulases will be examined and compared.

Cellulase specific oligonucleotide primers can be designed to locate cellulase sequences from microbially diverse environments without cultivation and mutagenesis. Students can also search publically-available metagenomic data sets for cellulase sequences to explore cellulase diversity from geographically distinct environments. Such sequences will provide useful information regarding the diversity and phylogeny of cellulase-like genes from a variety of environments. Finally research on the regulation of cellulase can be investigated on the isolated strains.

Different institutions will carry out these procedures from a variety of environments, such as marine sediments, estuaries, and agricultural soils. The strains, mutants, and sequences recovered will be a resource available to participating investigators and their students. A website will be maintained for easy access to data and current investigations. Students will also present their data at annual group meetings among the participating institutions.  

 
   
 


Project Materials:

Standard microbiological materials (including cellulase plate assay materials---carboxylmethylcellulose and Congo Red), 16S rRNA universal primers, PCR reagents and equipment, sequencing facilities, suicide transposon (such as pRL27c), cloning materials, BLAST and Ribosomal Databank Project access.  

 
 


Resources and References:

An introductory reference:

http://en.wikipedia.org/wiki/Cellulase

A useful series of articles on this subject:

Centeno MS, Goyal A, Prates JA, Ferreira LM, Gilbert HJ, Fontes CM. (2006). "Novel modular enzymes encoded by a cellulase gene cluster in Cellvibrio mixtus." FEMS Microbiol. Lett. 265: 26 - 34.

Percival, Zhang YH, Himmel, ME, and Mielenz, JR. (2006). "Outlook for cellulase improvement: screening and selection strategies." Biotechnol. Adv. 24: 452 - 481.

Hilden, L., Johansson, G. (2004). "Recent developments on cellulases and carbohydrate-binding modules with cellulose affinity." Biotechnol. Lett. 26: 1683 - 1693.

Lynd LR, Weimer PJ, van Zyl WH, and Pretorius IS. (2002). "Microbial cellulose utilization: fundamentals and biotechnology." Microbiol. Mol. Biol. Rev. 66: 506 - 577.

Beguin, P., Aubert, JP. (1994). The biological degradation of cellulose." FEMS Microbiol. Rev. 13: 25 - 58.  

 
   
 


Future Directions:

One advantage of this proposal is the "scalability" to different institutions. For example, it is possible to simply screen for aerobic heterotrophic cellulolytic microbes from diverse environments via plating and plate screening. This result demonstrates the diversity of the microbial world, as well as simple plate screens. Other institutions with more resources will be particularly interested in the transposon mutagenesis, mutant screening, and recovery of cellulase-specific sequences (random transposon insertions could also be analyzed if no cellulase deficient mutants are identified in a given screen). Finally, institutions or investigators solely interested in bioinformatics could use an in silico approach to analyze the cloned cellulase-specific sequences, and compare them to known cellulase genes---as well as to the 16S rRNA sequences recovered from each cellulase producing microbe.  

 
 


Attachments


- cellulase.ppt
- cellulase.pdf