Wisconsin Lutheran College’s 2013 iGEM team has been hard at work to create a secretion mechanism that is safe and customizable. Our way of approaching this is to make use of removable purification and secretion tags that are separated by a single enzyme cut site Nhel. This is the cut site that is the insertion point for any coding sequence of interest. Then the induced gene can be secreted from the cytoplasm to the extracellular space via a tripartite secI pump. In order to control a Tse2 toxin, a cymR-regulated T5cumate promoter is placed within the plasmid. This will prevent horizontal gene transfer. Essentially, if the plasmid is ejected from the bacteria and picked up by another, the gene producing the toxin will not be suppressed and will kill the cell. The plasmid also encodes a chimeric ompA-monoclonal antibody gene, which when expressed, inserts into the outer membrane. The antibody then selectively binds norovirus within the gut to hinder infection. This has great implications in the way of helping those with viruses in developing nations. This plasmid construct was used to over-express and secrete plant-based, polymer-degrading enzymes bglS, yesZ, and xynA. This will allow us to break down  hemicellulose and pectin. These enzymes were isolated from the cellulosome of Bacillus subtilis subtilis 168 to digest plant material. We utilized Escherichia coli Nissle 1917 containing a chromosome-integrated cymR gene to secrete these enzymes in the hope to accentuate dietary uptake for humans and livestock in impoverished countries.

Implications:

One of the great potential benefits of this project is provide impoverished people with more access to food. This project would allow a greater array of options for food sources, as well as increase the nutritional value of the food that is already present. The mechanism that this project provides will be extremely helpful to other iGEM teams. This system can be used to secrete any other enzyme or molecule chosen by an iGEM team or even general researchers.

Reception:

The intent of this project is to create a product that would aid developing countries, without necessarily changing their cultures, or “westernizing” them. In these countries, nutrition is a major concern. The lack of nutrition in developing countries has the added adverse consequence of increasing chronic diseases. “Nutrition is something easily changeable that has to power to prevent chronic disease, with scientific evidence increasingly supporting the view that alterations in diet have strong effects, both positive and negative, on health throughout life.” Nutrition is the first step to overall health, and therefor, our product could improve the general health of a developing country. Some sources even venture to say that food production is considered an evolutionary advance. Culture is not static, but dynamic. It is the aim of this project to enhance the cultures that it touches.Countries also have varying concerns about GMOs, especially the UK and India. Countries like the United States, Canada, and China would be more likely to accept these products. This product would go through further testing to ensure that it is safe for consumption in order to send to countries that could highly benefit from this product.

Future:

Polysaccharides are a convenient energy storage system for many organisms. In order to use the energy, the organism must break down the polysaccharide chain into smaller mono- and disaccharides. Cellulose is one such polysaccharide, an energy storage form for plants. Glucose monomers link together in long rigid chains to form cellulose. The plasmid breaks down plant products that are not cellulose, but similar in structure (hemicellulose and pectin). We have three enzymes to accomplish this: xynA, bglS, and yesZ. The endo-xylanase xynA cleaves xylan polysaccharide chains to form shorter xylan chains. The enzyme yesZ has beta-galactosidase (beta-galacturonidase) activity, cleaving exo-beta-D-galactans to produce single galactose molecules from RG I Pectin. The endo-beta-glucanase (bglS) will cleave internal 1,4 linkages adjacent to 1,3 linkages. A secretion system has been designed to release the enzymes from the bacteria. In future experiments, other enzyme-producing genes will be added to break down plant material further. Cellulase genes are first in line, as cellulose is the largest composition of cell wall. The 2008 Edinburgh team designed the genes required to break down cellulose into glucose. These are an exocellulase, an endocellulase, and a beta-glucosidase. Other enzymes required to fully degrade plant material would be added as well.

Overall, this project and the secretion system that it provides will be advantageous and beneficial to future iGEM teams as well as developing countries.