Team:Queens Canada/Project/Intro

C. elegans has been a long standing model organism for multicellular eukaryotes due to its simplistic structure and features which, despite being base in nature, provide highly analogous representations of biological processes in other model organisms. Generally, model organisms must have a set of common traits which allow for ease of use and observation such as rapid maturation and small growth cycles, small size, availability, and tractability. C. elegans is a particularly attractive model organism not only for its adherence to the above criteria but due to several other factors as well.

C. elegans is a transparent, nonparasitic nematode approximately 1mm in length and is found in most temperate soil climates. It is easily sustained in the lab through use of agar plates or liquid cultures at laboratory temperatures. It can feed solely on E. coli, and is hence cheaply cultivated. Its transparency allows for the study of cellular differentiation and tissue mapping via fluorescing proteins. The rapidity with which C.elegans reproduces and the large number of offspring generated per hermaphrodite leads to the production of high numbers of offspring in a short amount of time.

A population of C. elegans is comprised of two sexes: hermaphrodites and males, the former being predominant in any given population. Because of a lack of male-hermaphroditic mating, the genotypes of worms produced in culture remain generally homogenous. While the life span of a worm can be anywhere from 2-3 weeks in length, the worms themselves reach maturation within 3 days. At this point, all expression patterns in their cells are considered adult and the number of somatic cells within the worm remains at a constant of 959 for hermaphrodites and 1031 for males. The expected results of genetic construct microinjection can be detected and tested at this point.

The completely sequenced genome of C. elegans contributes to the organism’s status as a model for genetic engineering study, and as a result, means genetic manipulation techniques within the worm are well tested. Methods of gene manipulation by injection of plasmid vectors into the gonads are standard, as is RNAi gene knockout, for example. Furthermore, transgenes from higher organisms such as humans have been successfully expressed within C. elegans. This was proven with the insertion of human GPCRs (G-protein Coupled Receptors) into C. elegans where interactions between the human receptor and the worm G-protein were successful. These findings led QGEM 2011 to harness the potential of C. elegans through genetic engineering.

QGEM 2010 was the first iGEM team to introduce C. elegans as a chassis for genetic engineering in the iGEM competition. Their project focused on the creation of a C. elegans toolkit whereby other teams may easily perform feats of genetic engineering with the worm. This year, our focus was to build upon that concept, demonstrating the use of having a eukaryotic multicellular organism as a functional iGEM chassis and also a bioremediation tool, among other goals.

While the benefits of working with simpler prokaryotic organisms such as E. coli are not disputed, using eukaryotes as a chassis for genetic engineering has significant advantages as well. As a model eukaryotic organism, succesful manipulation of the C.elegans genome would quantify similar exploits using larger organisms. The portability between the genes of C. elegans and genes of other model organisms is high and, as is seen with the successful insertion of human GPCR transgenes into C. elegans, is proof that despite a largely divergent evolutionary pathways, eukaryotic systems share much in basic biological interactions. By utilizing a eukaryotic chassis, we show that other higher organisms may be manipulated similarly and that the possibilities of genetic engineering are truly endless. Already C. elegans has been instrumental in the study of aging and cancer development in humans.

G-protein Coupled receptors are seven-transmembrane domain receptors (also known as serpentine, 7TM, or G-protein-linked receptors) that bind ligands extracellularly, resulting in the activation of a signal transduction pathway within the cell upon ligand binding. They are sensory receptors which may bind lipids, peptides, light sensitive molecules, odour molecules, pheromones, hormones, ions, or neurotransmitters. GPCRs exist only in eukaryotic organisms where they affect many physiological processes including the visual and olefactory senses, behavioural and immune system regulation, autonomic nervous system transmission, and cell density sensing.

These receptors are integral membrane proteins with helical domains that span through the cell membrane. While the extracellular domain is available to bind an agonist or antagonist (ligand), the intracellular domain is coupled to a Guanine Nucleotide Binding Protein (G-Protein). The G-protein acts as a molecular switch, where the binding of GTP (guanosine triphosphate) represents the switch being “on” and the binding of GDP represents the switch being “off”.

The consequences of the switch being “on” or “off” relates to what occurs within the cell. The family of G-proteins which bind to GPCRs are termed Heterotrimeric G-proteins and are comprised of alpha, beta, and gamma subunits. In response to a conformational change in the GPCR, which results after a ligand binds to the GPCR, the G-protein exchanges a GDP for a GTP and then dissociates from the cell membrane to initiate a signal cascade within the cell, resulting in a biological response.

A GPCR is activated when a ligand binds to its extracellular domain. This activation induces a conformational change in the receptor and allows it to act as a guanine nucleotide exchange factor (GEF) which trades GDP for GTP on the Gα subunit of the G-protein. The Gα subunit may then dissociate from the Gβγ dimer, which anchors the G-protein to the membrane, and the GPCR to affect intracellular signalling proteins or target functional proteins directly depending on the α subunit type. Once dissociated, a new Gα subunit with bound GTP binds to the Gβγ subunit, and hence, signalling via GPCR may continue. The process is seen in the figure below. Two main signal transduction pathways are activated by GPCRs: the Cyclic adenosine monophosphate (cAMP) pathway or the Phosphatidylinositol Pathway, both of which act through other molecules to affect biological processes.

In C. elegans, a variety of behaviours are regulated by the chemosensory system where volatile and water soluble chemical signals in the environment are detected primarily through neurons in the amphid. These signals are associated with food, other organisms, or danger and can cause responses such as chemotaxis, changes in motility, rapid avoidance, and entry into or exit from the alternative dauer developmental stage. The amphid chemosensory organ of C.elegans is comprised of eleven neurons, all of which express a specific set GPCRs which bind distinct attractants, repellents, and pheromones. Within C. elegans, 500-1000 GPCRs are expressed in the amphid chemosensory neurons. Activation of these receptors triggers either the cGMP signal transduction pathway where cGMP acts as a secondary messenger which opens cGMP gated channels or the TRPV signal transduction pathway.

A table can be seen below which lists the GPCRs present in C.elegans the neurons in which they are expressed. It also shows the function of the GPCR and method of signal transduction.

QGEM 2011 focused on utilizing the chemosensory system of C.elegans by expressing non-native GPCRs in neurons where a forward chemotactic response was the ultimate downstream effect of ligand binding.