Metagenomics is a cultivation-independent approach for obtaining the genomic composition of microbial communities. Microbial communities are ubiquitous in nature. Microbes which are associated with the human body play important roles in human health and disease. These roles span from protecting us against infections from other bacteria, to being the causes of these diseases. A deeper understanding of these communities and how they function inside our bodies allows for advancements in treatments and preventions for these diseases. Recent developments in metagenomics have been driven by the emergence of Next-Generation Sequencing technologies and Third-Generation Sequencing technologies that have enabled cost-effective DNA sequencing and the generation of large volumes of genomic data. These technologies have allowed for the introduction of hybrid DNA assembly techniques to recover the genomes of the constituent microbes. While Next-Generation Sequencing technologies use paired-end sequencing reads from DNA fragments into short reads and have a relatively lower error rate, Third-Generation Sequencing technologies use much longer DNA fragments to generate longer reads, bringing contigs together for larger scaffolds with a higher error rate. Hybrid assemblers leverage both short and long read sequencing technologies and can be a critical step in the advancements of metagenomics, combining these technologies to allow for longer assemblies of DNA with lower error rates. We evaluate the strengths and weaknesses of the hybrid assembly framework using several state-of-the-art assemblers and simulated human microbiome datasets. Our work provides insights into metagenomic assembly and genome recovery, an important step towards a deeper understanding of the microbial communities that influence our well-being.

A primary aim of Synthetic Biology is the design and implementation of biological systems that perform engineered functions. However, the assembly of double-stranded DNA molecules is a major barrier to this progress, as it remains time consuming and laborious. Here I present three improved methods for DNA assembly. The first is based on, and makes use of, BioBricks. The second method relies on overlap-extension PCR to assemble non-standard parts. The third method improves upon overlap extension PCR by reducing the number of steps and the time it takes to assemble DNA. Finally, I show how the PCR-based assembly methods presented here can be used, in concert, with in vivo homologous recombination in yeast to assemble as many as 19 individual DNA parts in one step. These methods will also be used to assemble an incoherent feedforward loop, gene regulatory network.

A primary aim of Synthetic Biology is the design and implementation of biological systems that perform engineered functions. However, the assembly of double-stranded DNA molecules is a major barrier to this progress, as it remains time consuming and laborious. Here I present three improved methods for DNA assembly. The first is based on, and makes use of, BioBricks. The second method relies on overlap-extension PCR to assemble non-standard parts. The third method improves upon overlap extension PCR by reducing the number of steps and the time it takes to assemble DNA. Finally, I show how the PCR-based assembly methods presented here can be used, in concert, with in vivo homologous recombination in yeast to assemble as many as 19 individual DNA parts in one step. These methods will also be used to assemble an incoherent feedforward loop, gene regulatory network.

Cell-free transcription and translation systems promise to accelerate and simplify the engineering of synthetic proteins, biological circuits or metabolic pathways. Microfluidic droplet platforms can generate millions of reactions in parallel. This allows cell-free reactions to be miniaturized down to picoliter volumes. Nevertheless, the true potential of microfluidics have not been reached for cell-free bioengineering. Better approaches are needed for reaching sufficient in-drop expression levels while efficiently creating DNA diversity among droplets. This work develops a droplet microfluidic platform for single or multiple protein expression from a single DNA coated bead per droplet. This opens up the possibility to diversify a million droplets for synthetic biology applications.

Inexpensive petroleum is the cornerstone of the modern global economy despite its huge environmental costs and its nature as a non-renewable resource. While ninety percent of petroleum is ultimately used as fuel and can in principle be replaced by sources of renewable electricity, ten percent is used as a feedstock to produce societally important chemicals that cannot currently be made at a reasonable cost through alternative processes. In this dissertation, I will discuss my efforts, together with several colleagues, to apply synthetic biology approaches to the challenge of producing renewable petrochemical replacements. In Chapter 2, I discuss our efforts to engineer E. coli to produce fatty acids with a wide range of chain lengths at high yield, thereby providing an alternative platform for the production of diverse petrochemicals. In Chapter 3, I describe a novel method of DNA assembly that we developed to facilitate synthetic biology efforts such as those in Chapter 2. This method is capable of simultaneously assembling multiple DNA pieces with substantial sequence homology, a common challenge in synthetic biology. In Chapter 4, I discuss the development of a "bionic leaf": a hybrid microbial-inorganic catalyst that marries the advantages of photovoltaic-based light capture and microbial carbon fixation to achieve solar biomass yields greater than those observed in terrestrial plants. This technology offers a potentially low-cost alternative to photosynthesis as a source of biomass and derived chemicals and fuels. The work described in this dissertation demonstrates the capacity of synthetic biology to address the problem of renewable chemical production, and offers proof of principle demonstrations that both the scope and efficiency of biological chemical production may be improved.

DNA assembly and rational design are cornerstones of synthetic biology. While many DNA assembly standards have been published in recent years, only the Modular Cloning standard, or MoClo, has the advantage of publicly available part libraries for use in plant, yeast, and mammalian systems. No multipart modular library has previously been developed for use in prokaryotes. Building upon the existing MoClo assembly framework, we developed a collection of DNA parts and optimized MoClo protocols for use in E. coli. We present this assembly standard and library along with part characterization, design strategies, potential applications, and troubleshooting. Developed as part of the Cross-disciplinary Integration of Design Automation Research (CIDAR) lab collection of tools, the CIDAR MoClo Library is publicly available and contains promoters, ribosomal binding sites, coding sequences, terminators, vectors, and a set of fluorescent control plasmids. Optimized protocols reduce reaction time and cost by >80% from previously published protocols. The CIDAR MoClo Library is the first bacterial DNA part library compatible with a multipart assembly standard. To demonstrate the utility of the CIDAR MoClo system in a traditional biology context, we used the library and previous expression data to create a series of dual expression plasmids. In this manner, we produced a dual expression plasmid capable of expressing equimolar amounts of two variants of rabbit aldolase, a His-tagged wildtype protein and a single-amino-acid substitution mutant deficient in binding actin. This expression plasmid will enable the production of dimer-of-dimer heterotetramers needed for structural determination of the actin-aldolase interaction by electron microscopy. To employ CIDAR MoClo in a synthetic biology context, we produced a bioelectronic pH-mediated genetic logic gate with DNA circuits built using MoClo and integrated with Raspberry Pi computers, Twitter, and 3D printed components. Logic gates are an increasingly common biological tool with applications in cellular memory and biological computation. MoClo facilitates rapid iteration of genetic designs, better enabling the development of cellular logic. The CIDAR MoClo Library and assembly standard enable rapid design-build-test cycles in E. coli making this system advantageous for use in many areas of synthetic biology as well as traditional biological research.

Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON. / Doctor of Philosophy / Metabolic engineering is the use of genetic engineering to modify microorganisms in order to produce high yields of valuable commodity chemicals. The goal of this research is to develop new methods to improve genetic modification and selection of microbial cells. The specific objectives were to: (i) develop new tools for DNA assembly and manipulation, (ii) utilize small synthetic RNA to control protein expression level, (iii) use Raman spectroscopy to study phenotypic responses to environmental changes and (iv) enrich for microorganisms that detoxify dangerous toxins. First, a new technique for DNA assembly, named λ-PCR (lambda-PCR), was developed. This method allows the easy manipulation of plasmid DNA with high-efficiency and low-cost compared to traditional techniques. Second, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system in order to modulate (downregulate or overexpress) fluorescent protein expression. Next, Raman spectroscopy was used to assess phenotypic response of cyanobacterial cells to different environmental modifications (light settings, salts, sugar, etc…). Finally, genomic library was used to discover and characterize enzymes capable of degrading a mycotoxin.