Foundational technologies in synthetic biology : promoter measurement and peroxisome engineering
De Mora, Kim Stephen
Mora, Kim Stephen de
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The confluence of next generation DNA sequencing and synthesis when combined with the application of concepts such as standardization and modular design has led to the genesis of a new discipline. The nascent field of Synthetic Biology concerns the rational design and construction of genetic circuits, pathways, machines and eventually whole organisms. The immaturity of this field dictates that early research efforts, including this Thesis, describe foundational work towards the creation of tools which make biology more amenable to being engineered. The first part of this Thesis describes an attempt to standardize the measurement of transcriptional promoter activity in E. coli. A method to measure in vivo promoter activity was developed for E. coli and tested in a multi-institution trial. Comparable results were achieved with less than a two-fold range for the measured promoters across eight laboratories. A standardized measurement kit was created and distributed for use by the teams participating in the 2008 international Genetically Engineered Machines competition. Techniques learned measuring the activity of E. coli promoters were applied to a collection of S. cerevisiae strains. Several promoters were measured in synthetic dextrose media and ADH1 was measured in multiple media conditions. The outcome of these experiments is to consider proposing ADH1as the reference promoter in S. cerevisiae. The second aspect of this Thesis describes the construction of artificial organelles in S. cerevisiae. Artificial organelles hold the prospect of being able to insulate synthetic genetic pathways from the cell. Two proteins are essential for the biogenesis of the peroxisome organelle in humans and yeast, Pex3p and Pex19p. Pex3p functions as a peroxisomal membrane receptor for Pex19p, while Pex19p shuttles other peroxisomal proteins to the membrane, including Pex3p, creating a feedback loop. Human Pex19p has previously been shown to dock to yeast Pex3p and a version of yeast Pex19p has been shown to work with human Pex3p as a high degree of evolutionary conservation exists between these proteins. Because of these inter-species protein docking characteristics, there exists the possibility of creating bimodality: the ambition of the work was therefore to create a cell strain which possessed both synthetic “humanized” and natural yeast peroxisomes. An S. cerevisiae BY4741a derivative strain was engineered with fluorophore tagged versions of human (CFP) and yeast (YFP) Pex3p and untagged yeast and human Pex19p proteins. The results indicated the creation of a single population of peroxisomes when a measure of fluorescently imaged CFP and YFP peroxisomes were plotted on a scatter plot. A log of the ratio of CFP to YFP peroxisomes was plotted on a histogram and a normal distribution was found to best fit the curve, indicating a lack of bimodality. Finally, microscopy images of this strain were reviewed and by visual inspection, showed no evidence of distinct human or yeast peroxisomes. This experiment therefore produced no evidence of bimodality when examining the interactions of human and yeast Pex3p and Pex19p proteins. However, the four proteins were shown to interact closely to produce a single population of chimeric human-yeast peroxisomes. The peroxisome-deficient mutant phonotype strain was rescued using human Pex3p and Pex19p.