HOW WE DID IT
Dry Lab This year’s team extended last year’s project to find a higher quantity of DNA sequences and key enzymes within the cellulose and hemicellulose degradation pathway. The team focused on bioinformatics, more specifically a metagenomic pipeline and metagenomic library. The metagenomic library was produced through the collaboration with Dr. Trevor Charles at the University of Waterloo. Along with the production of the metagenomic pipeline and library, the team planned to co-culture cellulose-degrading E. coli and yeast in a bioreactor, in order to produce ethanol from cellulose. For the synthetic metagenomic library, the team used the Illumina MiSeq data from last year’s four fecal samples: Artic Wolf, Coyote, Porcupine, and Beaver. The porcupine Illumina MiSeq data was used to discover enzymes within the cellulose and hemicellulose degradation pathways. The process of shotgun sequencing, as seen in Illumina MiSeq technology, required short inputs of DNA that were ~300 base pairs. The length of base pairs required for a full gene is much longer. Thus, the team used another program, MegaHIT, which allowed them to stitch together sequences to get a fragment yield of 1,000 base pairs. Once the larger fragments were formed the team used the program, Prodigal, to identify the open reading frames (ORFs). Prodigal is responsible for locating ribosome binding sites, through the identification of the start and stop codons. Lastly, jackHMMR was used to find protein domains, with respect to the predicted function.
Wet Lab The team focused on beta-glucosidase, endoglucanase, and beta-xylanase for the cellulose and hemicellulose degradation pathway. The genes were optimized for expression within E. coli, and once all were modified, the team submitted them for synthesis at Integrated DNA Technologies (IDT). Each were cloned into the pET26b(+) expression vector system. pET26b(+) encodes a pelB sequence at the N-terminus of the protein of interest which is responsible for localization of the protein of interest to the periplasm. From the periplasm, soluble proteins are able to diffuse into the surrounding environment or are secreted. Once successfully cloned, the team aimed to characterize beta-xylanase. Beta-xylanase, is a key enzyme in the hemicellulose degradation pathway that cleaves xylose dimers to usable xylose monomers. Using a Coomassie Blue stain for total protein expression, the team was able to show that this novel enzyme is able to be expressed in E. coli in the pET26b(+) expression vector system. The team took a step further and attempted to assess the enzymatic activity of this novel beta-xylanase using a modified version of a cellulase/xylanase activity fluorophore assay (Chen et al., 2016). Using xylobiose conjugated to a fluoro-active molecule, we were able to determine the relative activity of our novel beta-xylanase relative to pet26b(+) alone. We discovered that under the conditions we provided the enzyme was not able to function properly (ie. cleave the substrate), possibly due to the differences in oxidation states of the gut and open air or due to pH differences from the protein’s natural environment.
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