Difference between revisions of "Team:Dalhousie/Description"

WHY

As fossil fuels continue to run out across the globe, many people are looking towards alternative sources of energy that are renewable and eco-friendlier. One of these options is biofuel, fuel made from organic matter. Biofuel is most commonly made from ethanol, or bioethanol, which can be used as a fuel for vehicles in its’ pure form. In the field of biofuel production, bioethanol made from cellulose continues to be the dominant form; however, harsh methods are required to be able to extract the sugars from cellulose and convert it to ethanol.
In Atlantic Canada, the main export is wood, pulp, and paper. The processes of these mills have been shown to be environmentally unsound with large quantities of hazardous waste being expelled. As many of our members have seen firsthand, growing up near these pulp and paper mills. Along with hazardous waste expulsion, the extraction of usable materials from wood is fairly inefficient. These mills leave behind a great deal of wood waste that could be used for biofuel production if broken down and converted to ethanol. The only question is: how?

WHAT

The Dalhousie iGEM team this year is focused on using the microbiome of the porcupine to solve this conversion of wood waste to ethanol.
A huge part of the porcupine’s diet is made from bark. Unable to digest the cellulose, hemi-cellulose, and lignin in the bark, the gut bacteria of the porcupine do the work instead. We hypothesized that the microbiome of the porcupine would contain enzymes that convert cellulose, hemi-cellulose, and lignin to glucose; a usable sugar.
We then hypothesized that if the genes coding for these enzymes were expressed in a vector in E. coli, the E. coli would then be able to digest cellulose and create glucose.
Finally, we hypothesized that a bioreactor system containing both E. coli and yeast would be able to create ethanol from wood waste as the yeast would ferment the glucose created by the E. coli.

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 metagenomic library, the team used the Illumina MiSeq data from last year’s four fecal samples: Artic Wolf, Coyote, Porcupine, and Beaver. 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 with endoglucanase, 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. (CONFIRMED SECRETION????)
Once successfully cloned, the enzymes were subjected to a number of assays for activity. Both beta-xylanase and beta-glucosidase’s activities were measured via a modified version of Chen et al. (2016)’s cellulase/xylanase activity fluorophore assay. Using glycosides (either cellobiose or xylobiose) conjugated to a fluoro-active molecule, we were able to determine the relative activity of our novel beta-glucosidase and beta-xylanase relative to pet26b alone. In addition, we assayed the enzymatic function of our beta-glucosidase and endoglucanase in 2 mechanisms related to growth on selective media. Beta-glucosidase containing BL21 DE3 E, coli were grown on M9 media plates containing cellobiose as the only carbon source. Thus, our E. coli could only grow on this selective media if they were expressing and utilizing the beta-glucosidase provided in the pET26b expression vector. NEED TO WRITE ABOUT ENDOGLUCANASE