Week 7. Semester 2. Research.

This week was spent doing research. I have concluded, with Matt's assistance and support that we will focus on isolating cellulases from fungi instead of anaerobic bacteria due to the difficulties we may face attempting to first, successfully grow anaerobic bacteria in the lab. And secondly, to extract cellulase complexes from bacteria. Because fungus secrete enzymes into their environment, they are a better candidate for this project because the exoenzymes would be present in the medium and could be isolated using the centrifuge and molecular filters (Manen et all. 2005). I am following Bethany's, a fellow S-STEM scholar, project closely because she is growing fungus for isolation and identification. Once she is able to find either Aspergillus or Trichoderma, the broth will be started.
The lab also purchased cellulase from Sigma in solution and a powder from from another supplier, so I also spent this week learning about cellulase and how it can be incorporated in my protocol. Hopefully after Spring break I will attempt an extraction using cellulase to digest the cell wall. There is still much research and experimentation I must do in order to carry out an effective extraction. Cellulase is the main component of plant cell walls and is a very rigid polysaccharide that maintains the cell shape of plant cells and provides additional structural support. As you may have read in my previous blogs, this is one of the main constraints to extracting high yields of DNA from plant material (Manen et all. 2005). Below are some pictures of cellulose that would hopefully aid to better describe its composition and toughness.

Colored transmission electron micrograph of cells in a young leaf (Robinson 2001).

Structure of Cellulose in a Plant Cell Wall (Moran-Mirabal 2003)

References

     

      Manen JF, Sinitsyna O, Aeschbach L, Markov AV, Sinitsyn A. 2005. A fully automatable enzymatic method for DNA extraction from plant tissues. BMC Plant Biology [Internet]; [cited Mar 12 2015]. Nov 3;5;23. Available from http://www.ncbi.nlm.nih.gov/pubmed/16269076.  Manen JF, Sinitsyna O, Aeschbach L, Markov AV, Sinitsyn A.

      Moran-Mirabal J. 2003. Advanced-microscopy techniques for the characterization of cellulose structure and cellulose-cellulase interactions. In: Cellulose - fundamental aspects [Internet]; [cited Mar 12 2015]. Available from: http://www.intechopen.com/books/cellulose-fundamental-aspects/advanced-microscopy-techniques-for-the-characterization-of-cellulose-structure-and-cellulose-cellula  

      Cosgrove DJ. 2001. Cell walls. In: Plant Sciences [Internet]; New York: Macmillan Reference USA; [cited Mar 12 2015]. Available from: http://ezproxy.pc.maricopa.edu:2048/login?url=http://go.galegroup.com/ps/i.do?id=GALE%7CCX3408000066&v=2.1&u=mcc_phoe&it=r&p=GVRL&sw=w&asid=75046d4a30a1cb609c50d446a88f427f