Each year, increasing numbers of individuals are added to organ wait lists worldwide. This is met with an ongoing shortage of donor organs, in part, limited by preservation technologies. A human heart can currently be stored for 6 hours before significant tissue damage results in a non-viable organ.
QGEM aimed to use antifreeze proteins (AFPs), natural proteins that enable certain organisms (such as the ocean pout shown above) to survive in sub-zero climates, to rectify this limitation. We have engineered two classes of AFPs focused on improving protein function and stability, respectively. Our primary objective has been the development of an anchoring system to increase local concentration of AFPs by attachment to a self-assembling scaffold. This system increases the probability of favorable interaction with ice surfaces thereby improving AFP activity. Our secondary project structurally modified an AFP, enabling the protein to withstand a more diverse chemical environment. This increases potential industrial applications in food and energy sectors.
Found across all domains of life, inteins are protein elements capable of autocatalytic splicing. This phenomenon, termed protein splicing, allows inteins to be a point of control in forming mature and functional protein products. Current genetic circuits are limited by transcriptional lag, rendering them limited in applications which require fidelity and responsiveness.
Inteins represent an innovative solution to improving the sensitivity of biosensors and synthetic circuits, circumventing this lag and controlling the output of the circuit at the protein level. Here, we have created an intein toolkit, designed to introduce and facilitate the use of inteins in future iGEM projects. We have engineered several inducible intein switches capable of being activated by common environmental triggers as well as small molecules. To showcase the utility of inteins, we have also developed an intein-based system to tackle mitochondrial disease by allotropically expressing and transporting mitochondrial proteins using inteins.
Biosynthesis and Breakdown of Human Odour Compounds for the Behavioural Manipulation of Malarial Mosquitoes
Malarial mosquitoes are developing resistance to key insecticides and drugs, and are becoming diurnal to avoid treated mosquito nets. Recent studies have shown that the African mosquito uses human foot odour to locate its host, a trait that is enhanced when the insect is carrying malaria. We planned to combine a carboxylic acid reductase with an acetyl transferase in order to create E. coli capable of converting a major component of foot odour (isovaleric acid) into banana odour (isoamyl ester). This could have both commercial and humanitarian applications.
Our second goal was to deliberately synthesize mosquito attractants inside traps. Recent research has shown that a mixture of carbon dioxide and foot odour volatiles can be more attractive than a human. We have chosen indole as our first attractant, a compound naturally found in human sweat. We hope our project will show that bioremediation and biosynthesis techniques have applications in mosquito control.
This year, Queen’s iGEM team used flagella to host heterologous proteins that result in thousands of useful enzymes organized in an extensive scaffold, with the benefits of extracellular synthesis, degradation, and arrangement. The fliC (flagellin) protein is known to spontaneously polymerize to form the length of flagella in E.coli.
By replacing the variable D3 domain of the fliC protein with proteins for binding, degradation, adhesion, and synthesis, we can increase the efficiency of bioremediation and biosynthesis, and facilitate the collection of products in situ or ex situ. Future applications for engineering the flagellin protein include attaching heavy-metal binding proteins to bacterial flagella, a potential new bioremediation solution to heavy-metal pollution by the Alberta oil sand industry.
Engineering C. elegans for advanced bioremediation
Naphthalene is a pollutant produced by oil sands operations. The Queen’s team has engineered the nematode worm C. elegans into a toolkit for dealing with this compound in the soil. We have produced constructs with GPCRs from M. musculus, R. norvegicus, and H. sapiens intended to enhance the worm’s ability to chemotax toward naphthalene. We worked on a field bioassay based on fluorescent proteins that indicates the presence of naphthalene in a soil sample.
The goal was to have a population of green fluorescent worms chemotaxing toward and a population of red fluorescent worms chemotaxing away from the napthalene in the soil sample. Finally, we have biobricked the P. putida gene nahD, which encodes a degradative enzyme as part of a naphthalene catabolic pathway. The nahD gene encodes the enzyme 2-hydroxychromene-2-carboxylic acid isomerase, which catalyzes the fourth step in the catabolic pathway.
Engineering C. elegans for a powerful iGEM toolkit for multicellular organism projects
Historically, the iGEM competition has tended away from working with eukaryotic and multicellular organisms, limiting prospects for higher levels of project complexity in favor of simpler and easier-to-understand bacteria. The nematode worm Caenorhabditis elegans was examined as a prospective chassis for use in the competition. Once it was decided that the opportunities presented by the organism appeared to outweigh the challenges involved in working with it, a foundational library of parts was built and tested within the organism. This collection includes useful promoters, reporters, effectors, and a terminator. An educational resource specifically targeted at iGEM participants was written and incorporated into the team wiki in order to assist future teams in learning about and exploring the possibilities offered by C. elegans.