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Team:WLC-Milwaukee/Project
From 2013.igem.org
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Overall project
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Project Details
WHAT: We are repurposing cellulolytic genes to increase bioavailable energy by breaking down cellulose into glucose, which can be used for humanitarian efforts, economic mobility and Industrial application
How it will be done: Pick organism of delivery E. coli Nissle 1917 https://2012.igem.org/Team:Penn/Nissle (previous iGEM team discusses why they used E. coli Nissle 1917, public opinion as a therapeutic system - "more palatable to the general public," use the word "probiotic," colonizes borders between live and necrotic tissue, among other positives) http://jmm.sgmjournals.org/content/59/4/392.full.pdf (Nissle 1917 outcompetes intestinal pathogens, why it is good as a probiotic, used as probiotic remedy since the 1920's in Europe) http://www.ncbi.nlm.nih.gov/pubmed/18240278 (abstract, clinical use in ibd, one of the best-studied probiotic strains, potential to protect from other bacterial infections in the gastrointestinal tract, lack of virulence, safe and effective candidate in the treatment of ibd) http://informahealthcare.com/doi/abs/10.3109/08910600903444267 (non-pathogenic, carries no pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, noninvasive, not uropathogenic, rapidly killed by non-specific defense factors of blood serum. . ., fitness factors: genes for the production of microcins which inhibit the growth of other enterobacteria, special lipopolysaccharide in the outer cell membrane which causes the immunomodulating properties without immunotoxic effects, strengthens epithelial barrier) Pick organism of primary testing E. coli lab strain K12 http://ecoliwiki.net/colipedia/index.php/Category:Strain:E._coli_K-12 (I am assuming wildtype, there are many different lab strains from e. coli K-12.) this strain is a commonly used strain and well documented Pick organism to derive cellulase genes from Bacillus Subtilis Pick cellulolytic genes xynA yesZ bglS eglS Design delivery system We will use the Washington university 2009 gram negative sec I secretion pump utilizing the pump found on part BBa_K215107. Our genes of interest will be added to the part BBa_K21500 which tags the proteins for secretion by part BBa_K215107 Design Primers for xynA, yesZ, bglS, and eglS using Washington University supplied instructions
Isolate Genomic DNA from B. subtilis lab strains and cellulolytic bacteria that can be found in composting plant matter Perform PCR with designed primers using genomic DNA from different strain. Characterize, isolate and insert PCR product into part BBa_K215002 Transform BBa_K215002 with different cellulolytic gens and part BBa_K215107 into E.coli Preform Western/southern/qPCR to confirm transcription of our cellulolytic genes Test cellulolytic function with cellulose plates Test cellulolytic enzymes in simulated gut conditions Transform E.coli Nissle 1917 Test on mice!
• Economic mobility o The cellulolytic probiotic strain that we are designing would be ideal for providing increased economic benefit and mobility to the underpriviledged populations that are the target individuals for this product. This would increase their economic powere by increasing the bioavailable resources that were once unavailable. This would do so by increasing nutrition in the cattle thus increasing the market value of the livestock, increasing the revenue available per capita. Also the increase in caloric availability could also apply to carbohydrate food sources thus allowing for an increased self sufficiency and, hopefully, the formation of a surplus that could enter into the world market. It is this entry into the world market that could facilitate a upward movement of under developed countries. • Industrial application o The use of the cellulolytic E coli nissle 1917 would increase the availability of glucose for industrial application by opening a new avenue for using the renewable and abundant environmental cellulose. This could lead to an increased industrial self sufficiency by decreasing dependence on non renewable resources (i.e fossil fuels) or repurposed resources (i.e. corn and rice). In the case of fossil fuel, the world market depends on it due to its high demand but with the increased amount of bioavailable glucose there would be an increased availability of ethanol for use as a renewable fuel source. In addition with an increase in bioavailable glucose the world will be able to move away from high fructose corn syrup, which has been linked to an increased obesity rate in America, and towards high glucose syrup. This would mean a decreased reliance on corn allowing its use in other industrial or economic applications. All in all the use of a cellulolytic bacteria in industry would allow for a repurposing of what was once considered “trash” and instead turn it into a potential “treasure” using it as a resource, recycling what was once useless into something that can now be considered a reliable and renewable resource. • Humanitarian efforts o Through the use of a known probiotic strain E. Coli Nissile 1917 the cellulolytic genes studied will be able to be used safely and effectively in human subjects. The effect of the cellulolytic probiotic strain would be to increase the caloric intake in the individuals who live as sustenance farmers, refugees, and in areas that do not provide enough agricultural means so as to provide sufficient calories for the population housed in the area. This increase in caloric intake would mean an increase in self sufficiency in third world countries and a decrease in economic strain for the supporting nations. The increase in self sufficiency would mean an increase in economic surplus within the country in question, eventually leading to an increase in the rate of development in these countries.
