Date of Award:

5-2011

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biological and Irrigation Engineering

Advisor/Chair:

H. Scott Hinton

Abstract

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.

Comments

This work made publicly available electronically on April 11, 2011.

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