Team:Lethbridge HS/Description

Project

How does our project work?

• Description

• What is nuclease? What is dextranase?

• What we are doing differently

• Extracellular Polymeric Substance Matrix

Overview

Without the hard work of bees, the difficulty of planning dinner would increase significantly. Taking time out of our daily routine to consider all that bees have done for us would take quite a while. We would not have some of the grains, fabrics, fruits, and vegetables that we enjoy. Alfalfa, cotton, almonds, oranges, cherries, and livestock that feed on bee pollinated crops would all decline significantly, if not completely perish. We would not have the flowers and trees that make our world beautifully diverse. Bees pollinate 70 out of the top 100 food crops, which supply 90% of the world’s nutrition. It is apparent that bees are an integral part of the ecosystem and human life.

Unfortunately, bees have been in decline for about 30 years and the death rate has gone up in the past decade. This phenomenon is caused by a number of factors, including the use of neonicotinoid insecticides, climate change, and parasites. One of the most notable parasitic mites that infects bees is the Varroa destructor mite. This particular mite feeds off of the bee’s hemolymph, the blood-like substance, transmitting viruses like Deformed Wing Virus (DWV) in the process. DWV disrupts the bee’s development, causing them to have stubby wings and short bodies that prevent flight.

A few methods to deal with V. destructor are ineffective as they do not target the mite at a crucial phase when the bee is most susceptible. The larvae are sealed into combs with the mites, a place that some treatments simply can not reach.Other treatments rely on chemicals that increase bee mortality rate. For example, formic acid has a harmful effect on capped and uncapped honey bee brood1. In addition, resistance against various methods are developing in certain Varroa mite populations.

Currents techniques used to deal with V. destructor mites include using bio-pesticides, alcohol washes, synthetic chemical pesticides and the sugar shake method.

Our modus operandi: protect all the bees and larvae from the inside out. Using synthetic biology, we designed E.coli that produces the miticide oxalic acid in the midgut of any bee that ingests it. Our construct will include the Petal Death Protein (PDP). This protein aids in the conversion of oxaloacetate, a natural product of the Kreb’s cycle, into oxalic acid. Oxalic acid is an optimal weapon against the mites as the concentration needed to kill a mite is 70 times less lethal to the bee. The acid is already being used in syrup and spray solutions.

In order to apply this construct to the hives, we plan to use nurse bees to feed a mixture of E.coli and sucrose to the young bees. In the midgut the E.coli will produce low concentrations of oxalic acid that are lethal to only the mite, and that will diffuse into the hemolymph of the bee. This will cause the Varroa destructor to die if it sucks the bee’s hemolymph.

Our construct is more effective than current methods used, as it allows us to tackle the issue at an earlier stage, when the bee has not hatched from the uncapped brood. It also targets the mite more directly than other spray-on insecticides. With this project, we will take one step closer to combatting Colony Collapse Disorder (CCD), the devastating issue of decreasing global bee population, by improving the method of killing one of the main culprits of CCD: the Varroa destructor mite.

Description The purpose of hospitals is to help people get better. However, in the United States, 2 million people are infected during their hospital stay and bacterial biofilms are responsible of 65% ( Sydnor, E., & Perl, T. (2011, January 24). Hospital Epidemiology and Infection Control in Acute-Care Settings. Retrieved September 18, 2015, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3021207/) of all hospital acquired infections. A biofilm is a conglomeration of bacteria that is enclosed in a matrix of sugars and extracellular DNA, this helps to hold the bacteria together like a community. Biofilms can adhere to any surface and are commonly found in nature. However, biofilms can become problematic when they adhere to surgical tools such as catheters, endotracheal tubes, and scalpels. Currents methods used to destroy biofilms include antibiotics and biocides. These methods are often expensive, harsh, and ineffective; biofilms are notorious for developing resistance to chemical treatments. Instead of trying to kill the bacteria in the biofilm, we decided to degrade the matrix that protects it. We have created a cocktail of Nuclease and Dextranase to achieve this purpose. Once the matrix is degraded, the bacteria inside can be eliminated without the use of expensive chemicals. What is nuclease? What is dextranase? As mentioned earlier, one of the two targets for dispersing the biofilm is its extracellular DNA. To do so, we are using E.coli to secrete nuclease. A nuclease is an enzyme that catalyzes the hydrolysis of the phosphodiester bonds of nucleic acids. More specifically, the nuclease utilized by our team is a Micrococcal nuclease (Mnase) capable of cleaving single-stranded and double-stranded nucleic acids. The irony of the situation being that the nuclease bio brick (Part: BBa_K729004) being used is derived from Staphylococcus aureus. S. aureus biofilms are a major problem on medical equipment, and account for many infections. The S. aureus biofilm uses the Mnase for partial dispersal of its outer layer, so its inner colonies can spread outside and grow more biofilms. S aureus. Being a gram positive, ubiquitous bacteria, has one of the toughest biofilms. And hence, the strength of the Mnase is equally supplementary. Although the Mnase is derived from S. aureus, the function and the effect of the enzyme is not limited to that bacteria. Since extracellular DNA is component of almost every biofilm, the effect of the enzyme will not be affected by a change in the species/strain of bacteria. To further enhance the effects of Mnase, it will be applied in a mixture that also includes Dextranase. Dextranase is an enzyme that catalyzes the hydrolysis of bonds within dextran. Dextran is a component of the exopolysaccharide (EPS) matrix, common to many biofilms. The dextranase used for the construct, is an alpha-dextranase derived from Chaetomium gracile (a dematiaceous mold from the fungi family). Dextranase, by degrading parts of the EPS matrix would allow for Mnase to further seep into the biofilm, and thus increase the overall efficiency of the mixture. What we are doing differently. Past projects have targeted the bonds within the biofilm structure to disperse the biofilms, but since there is a variety of major bonds found within different biofilms, the effects of the construct have been limited to some bacterial species. But, by targeting the extracellular DNA and the exopolysaccharide matrix (common components of almost every biofilm), our aim is to create a general all-purpose dispersant, capable of working on a variety of biofilms, thriving in a variety of settings. Extracellular Polymeric Substance Matrix: Sticky Stuff! The bacteria in the biofilm are surrounded by an extracellular polymeric substance (EPS) matrix. This matrix is comprised of water, which hydrates the cells; various sugars, to provide nutrients and the sticky structure of the biofilm; proteins, which are typically enzymes; lipids; and extracellular DNA, which serves as a structural component of the biofilms. The EPS matrix constitutes 50-90% of a biofilm’s organic matter. The purpose of the EPS matrix is to adhere the bacteria to a surface and protect the biofilm against any harsh environmental conditions. The matrix also provides a “transport system” so that nutrients, water, and enzymes can move around the structure to meet the needs of each cell. The production of the EPS matrix is, in part, regulated by quorum sensing; this is a way for bacteria to communicate with each other via chemical signalling molecules. Like a “quorum”, once there are enough bacteria, the bacteria are able to communicate with each other to collectively express a gene, in this is case they would be producing the EPS.

Chen, C., Sun, Q., Narayanan, B., Nuss, D., & Herzberg, O. (2010). Structure of Oxalacetate Acetylhydrolase, a Virulence Factor of the Chestnut Blight Fungus. Journal of Biological Chemistry, 26685-26696.

MetaCyc Pathway: Oxalate biosynthesis. (n.d.). Retrieved September 19, 2015, from http://vm-trypanocyc.toulouse.inra.fr/META/NEW-IMAGE?type=PATHWAY&object=PWY-6699

Oliver, R (2006). Oxalic Acid: Questions, Answers and More Questions: Part 1 of 2 Parts. Scientific beekeeping. Retrieved from http://scientificbeekeeping.com/oxalic-acid-questions-answers-and-more-questions-part-1-of-2-parts/.

Underwood, R. M., & Currie, R. W. (2006) The effects of temperature and dose of formic acid on treatment efficacy against Varroa destructor (Acari: Varroidae), a parasite of Apis mellifera (Hymenoptera: Apidae).

Experiment & Applied Acarology, March 2003, Volume 29, Issue 3, pp 303-313. http://link.springer.com/article/10.1023/A:1025892906393#page-1

Tarpy, D. R., & Summers, J. (n.d.). Managing Varroa Mites in Honey Bee Colonies. In NCSU Apiculture Program. Retrieved September 18, 2015, from http://www.cals.ncsu.edu/entomology/apiculture/pdfs/2.03%20copy.pdf

Save the Bees (n.d.). In GreenPeace. Retrieved September 18, 2015, from http://www.greenpeace.org/usa/sustainable- agriculture/save-the-bees/