Our Project- Sugar Rush

Malnourishment has been a problem in the world for as long as history has been recorded. Many of the complex sugars found in plants cannot be broken down by humans or even the ruminants of cattle or goats. By breaking these complex sugars down into digestible sugars, the caloric value of these foods could be increased. Through these means we hope to make a large stride toward lessening the world’s caloric deficit.

Our Plasmid

Our final plasmid will consist of four main components: the pump, the secretion tag, the safety mechanism, and the antinorovirus particle antibody. The pump (K215104) is a type I Erwinia chrysanthemi secretion system. This system works in conjunction with the prtB secretion tag (K215002) so only proteins containing the prtB C-terminal tag are secreted. This tag is built into K215104, which consists of a pLAC promoter, strong RBS site, and signal and streptavidin binding tags. These tags include a Nano-tag, a 6x His tag, a TEV cut site, an Nhe1 site, and a 181 C-terminal amino acid strand. This strand must be present in order for the secretion tag to signal the secretion of the protein of interest, which can be inserted at the NheI site. This site allows the system to be customizable; though in our system, we are focused on secreting three specific enzymes: yesZ, xynA, and bglS. These enzymes target specific types of biomatter which, when broken down, can be utilized for caloric content.

The safety mechanism of our plasmid has a T5 cumate operator, which is a suicide gene that arrests growth when expressed intracellularly. The T5 cumate operator is suppressed by CymR, meaning that the plasmid will only work in a bacterium that natively expresses CymR. Without CymR expression, the T5 cumate operator becomes active and expresses Tse2 toxin, which causes the death of the cell. Another facet of the safety mechanism is that p-cumate, when introduced into the system, can inhibit CymR. This will once again cause the expression of the Tse2 toxin. Only in a system where CymR is expressed and p-cumate is absent will the T5 cumate operator be repressed. The cell will then survive.

Our plasmid also contains an antinorovirus particle antibody (K875004) in fusion of LPP-OmpA. Once expressed, the antinorovirus monocolonal antibody binds to the C-terminus of the chimeric protein LPP-OmpA. The protein then introduces itself into the outer membrane, thus displaying the antibody extracellularly. The antibody then binds to the native, recombinant norovirus particles. This inhibits the virus’s interaction with the cells.

Enzymes

We've isolated three enzymes from Bacillus subtilis subtilis 168 for use in this project: yesZ, xynA, and bglS. All three of these enzymes play an individual role in the breakdown of biomatter in a digestive system. YesZ cleaves RG I Pectin, which is a primary component in the cell wall of plants. Pectin is cleaved into single galactose molecules that can be metabolized by both human and animal digestive systems. XynA degrades xylan, a polysaccharide chain that cannot be broken down by the human body. Xylan is a major structural component of plant cell walls, and, due to crosslinks with cellulose and other cell wall components, inhibits the access of cellulases. BglS is a beta-glucanase that breaks down beta-glucan. BglS breaks glycosidic bonds within beta-glucan and hydrolyzes these glucans making the substance more easily digestible.

Antinorovirus Particle

In addition to caloric deficit, norovirus outbreaks frequent our project’s target populations. The lower quality of living and increased water contamination allows easy transmission throughout these developing nations. Infected individuals experience gastroenteritis and severe dehydration, at worse, leading to death.

Our plasmid includes an antinorovirus particle antibody in fusion with LPP-OmpA. Once expressed, the antinorovirus monocolonal antibody binds to the C-terminus of the chimeric protein LPP-OmpA. The protein introduces itself into the outer membrane, thus displaying the antibody extracellularly. The antibody then binds to native, recombinant norovirus particles. This inhibits the virus’s interaction with the infected individual's cells, which ultimately allows the organism that ingested the probiotic to successfully combat the virus.

Safety Mechanism

Our final plasmid has a portion dedicated to anti-horizontal transfer, preventing the DNA from escaping into the wild or being misused. This section, referred to as “Safety,” is compromised of the Tse2 intracellular toxin gene (BBa_K314200) downstream of a T5 Cumate Operator (BBa_K875001). The molecule CymR, which is natively expressed in E. coli Nissle 1917, represses this operator. Bacteria that are transformed by this plasmid that will manufacture the toxin and self-destruct. In addition, the CymR repressor can be inactivated by another molecule, p-cumate. The combination of these two properties allows our DNA to be used in a controlled fashion, allowing for cheap treatment of both intentional or unintentional outbreaks.

The target countries of our project hold cultural practices that differ greatly from our own. Because of this, our team strives to be culturally sensitive and uphold the cultural practices of the indigenous people. Others that have attempted to help these natives have unknowingly invaded the traditional cultural practices. While these attempts did increase the natives’ quality of life, they often times, destroyed the portion of their lives that defined the culture. We are attempting to introduce a product that doesn’t interfere with the indigenous culture, while simultaneously positively affecting their quality of life. Our project will allow farmers to tend to their herds, but allow them to use fewer resources to sustain them.

We have also investigated the key role education plays in effectively improving quality of life of others. Education is vital in the assistance of different countries. This includes the education of the indigenous individuals, but also the non-native peoples. Our team took it upon ourselves to help educate future scientists in the subject of biotechnology by hosting a week long summer camp for high school students. During this camp we focused on introducing students to the basics of cellular biology and biotechnology’s potential to solve grand world problems.