The concept of introducing engineering principles of abstraction and standardization into synthetic biology has received increasing attention in the past several years and continues to be in the forefront of synthetic biology. One direction being pursued by synthetic biologists is creation of modular biological parts (BioBrickTM) that can be readily synthesized and mixed together in different combinations. However, most standard BioBrickTM parts in the Registry were designed for E. coli, although synthesis of specific BioBrickTM parts for other bacteria, such as for yeast and cyanobacteria, have begun. Besides, at the present time, there are only three chassis, which include E. coli, Bacillus subtilis, and a cell-free chassis, available in the Registry. Thus, the choices of BioBrickTM chassis are very limited. In addition, most BioBrickTM parts in the Registry have not been characterized. In the present study, the BioBrickTM concept was extended to the photosynthetic bacteria, Rhodobacter sphaeroides. In order to do that, a BioBrickTM compatible gene expression system was designed to convert R. sphaeroides to potential solar powered bio-factories or bio-refineries. This gene expression system was composed of BioBrickTM promoters, Ribosome Binding Sites (RBSs), and terminators in a BioBrickTM compatible cloning vector and its function has been validated through the expression of fluorescent proteins. In addition, a bioluminescence-based BioBrickTM characterization method was developed in this study. This method was based on a cloning vector that includes two adjacent operons, with each expressing a different luciferase reporter gene. The measured optical signals from the two expressed bioluminescent reporters were then used to predict the performance of promoters, RBSs, and terminators. Based on this bioluminescence-based BioBrickTM characterization method, two BioBrickTM characterizations kits, one for E. coli and one for R. sphaeroides, were developed. BioBrickTM parts that include seven promoters, six RBSs, and six terminators were characterized using the E. coli characterization kit. R. sphaeroides BioBrickTM parts were characterized when R. sphaeroides containing the BioBrickTM measurement constructs were cultured by both anaerobic photosynthesis and by aerobic respiration respectively. The experimental results showed that the activities of these R. sphaeroides BioBrickTM parts were very similar for the cells growing under two different conditions.

Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen known to cause a variety of infections that are difficult to treat due to extremely high resistance to almost all antibiotics currently in clinical use. One of the major contributors to this resistance is the active efflux of antibiotics from the cell, primarily by action of the Resistance Nodulation Division (RND) family of efflux pumps. These pumps are composed of three proteins; an inner membrane RND pump, a periplasmic membrane fusion adaptor protein, and an outer membrane protein. The mechanism by which the three proteins interact to form a functional complex is largely unknown and the methods currently available for their study involves expression systems geared for high levels of expression. In the case of membrane proteins which play a role in clinically relevant activities, such as multidrug resistance, an expression system which does not always reflect biologically relevant levels of protein in the cell is not ideal for studying their interactions as correlation of conclusions from interaction studies to true interactions may not be possible. In this study a single copy gene expression system was designed and demonstrated to better reflect clinically relevant levels of overexpression compared to a multi-copy expression system. Quantitative-real time PCR analysis of C-terminally hexa-histidine tagged outermembrane protein, OpmH, expression shows approximately 100-fold and 20-fold overexpression from multi-copy and single-copy expression systems respectively. OpmH-H6 was successfully purified from both multi copy and single copy expression systems with proportionate purification schemes indicating the feasibility of single copy expression systems for the study of membrane bound protein complexes. / UOIT

La structuration des réseaux neuronaux est un processus fondamental qui assure le bon fonctionnement du cerveau. Afin de comprendre la formation et l’activité de ces réseaux, nous souhaitons développer une méthode qui permette de contrôler in vivo sous l’action de la lumière l'expression de gènes ciblés impliqués dans ce phénomène,à l’échelle d’une cellule neuronale individuelle. Pour ce faire, nous utilisons une réaction de photo-clivage permettant de libérer de façon contrôlée un inducteur d’expression de gène sous l’action de la lumière, à l’aide de groupements photo-labiles sensibles aux excitations bi-photoniques développés au laboratoire et favorables aux applications in vivo. Afin de photo-réguler l'expression des gènes in vivo et avec un contrôle spatiotemporel élevé, nous combinons le système d’expression de gène inductible par la tétracycline « Tet-on » à une variété de précurseurs photo-activables d’analogues de tétracycline que nous avons synthétisés. Ceci devrait nous permettre de disposer d’un système efficace pour l’expression in vivo d’un gène d’intérêt par excitation lumineuse,et plus précisément dans le but de photo-réguler le gène Kir2.1 impliqué dans la régulation de l’activité électrique des neurones. / The structural neural network’s is a fundamental process that ensures the proper functioning of the brain. To understand the formation and activity of these networks, we are developing a method which spatio-temporally controlled in vivo, the expression of targeted genes involved in this process at individual neuron cells scale by light. To achieve this in vivo tests, it is necessary to work with methods which are orthogonal to their cellular environment. Photochemical activation by photo-cleavage of an inert biological precursor offers a unique orthogonal way to attain this spatio-temporal control. Therefore, we have recently developed a new family of photoremovable group which are sensitive to two-photon (TP) excitation sensitive, in order to irradiate at favorable wave-lengths for in vivo applications. Moreover, to photo-regulate the expression of genes with high spatial and temporal resolution, we are combining the inducible gene expression system by tetracycline called « Tet-on » system to different photo-activable precursors of tetracycline analogs obtained by hemi-synthesis. All this, should allow us to get an effective system for the in vivo expression of a gene of interest by light excitation in order to photoactivate Kir2.1, a gene that cell autonomously silences the electrical activity of neurons in a subset of cells.