The emerging field of synthetic biology evolved from biological research much the same way synthetic chemistry evolved from chemical research; with accumulated knowledge of the structure of single genes and proteins and the methodologies to manipulate them, researchers turn to forward engineer complex biological systems to effectively manipulate living systems. Much like in the case of enzyme engineering, a rationally designed biological network is currently beyond our reach, and we turn to directed evolution to circumvent this gap in knowledge. Yet the unique nature of live biological networks uncovered new challenges previously unmet by single-gene molecular technologies, and extrapolation of current technologies to the manipulation of multi-component has proven laborious and inefficient. To establish engineering technologies for living cells, novel directed evolution techniques are sought for that are compatible with simultaneous manipulation of multiple biological components in vivo. In this work, we explore techniques for library DNA mutagenesis in the context of single and multiple genes. Chapter 1 provides an overview of the challenges in expanding current in vivo directed evolution methods from single enzymes, to the design pathways and cells. Chapter 2 describes the design and characterization of an assay for combinatorial directed evolution of a single metabolic enzyme. In Chapter 3 we present the utilization of our DNA assembly system, Reiterative Recombination, for attenuation of metabolic pathways. We use a library of promoters to combinatorially vary the expression of genes in the heterologous lycopene biosynthetic pathway in S. cerevisiae. Finally, Chapter 4 explores the calibration of the dynamic range of genetic selection, using metabolic enzyme activity as probe for cell survival.

This document provides background material in support of the 2005 intercollegiate Genetically Engineered Machine Competition (aka iGEM). Herein, you can read quick summaries of earlier competitions and courses, obtain a general view of the work from both a technical and educational perspective, and read about our current understanding of how to engineering biology (e.g., PoPS, abstraction, and so on). / MIT iCampus

Why do biological risks exist? Can we develop and implement a strategy for thoughtfully approaching future biological risks? This short, working report provides an abstract introduction to the problem of biological risk and outlines how technical and societal approaches should be combined in order to best address the challenge.

Draft public declaration from the Second International Meeting on Synthetic Biology (May 20-22, 2006, Berkeley, CA)

Heterologous biosynthetic pathway expression is an essential tool for natural products biochemists. It has provided a powerful methodology for elucidating and characterizing bacterial biosynthetic pathways. In this thesis I will discuss methods to harness biosynthetic pathways for the heterologous production of a monosaccharide natural product, Legionaminic acid (Leg5,7Ac2). This carbohydrate belongs to a family of sugars called nonulosonic acids (nine carbon α-keto acids) and is a 5,7-diamino derivative of sialic acid (Neu5Ac). It is found in cell surface glycoconjugates of bacteria including pathogens such as Helicobacter pylori, Campylobacter jejuni, Acinetobacter baumanii and Legionella pneumophila. Their presence on bacteria has been correlated with virulence in humans by mechanisms that likely involve subversion of the host’s immune system or interactions with host cell surfaces due to its similarity to sialic acid. Further investigation into their role in bacterial physiology and pathogenicity is limited as there are no effective methods to produce sufficient quantities of these carbohydrates. Herein, I harness microbial biosynthetic pathways via metabolic and genetic engineering to produce these complex nonulosonic acids. Leg5,7Ac2 is produced from N-acetylglucosamine using the Escherichia coli strain BRL04, which results in substantial over-production (> 100 mg L-1 of culture). Pure Leg5,7Ac2 could be readily isolated and converted into CMP-activated Leg5,7Ac2 for biochemical applications as well as the phenyl thioglycoside for chemical synthesis applications. A similar strategy was employed to access the related nonulosonic acid pseudaminic acid (Pse5,7Ac2). A biosynthetic pathway for production of Pse5,7Ac2 was constructed from H. pylori and C. jejuni and expressed in E. coli BRL04. Unlike Leg5,7Ac2, Pse5,7Ac2 was produced in low yields (< 20 mg L-1). A number of modifications were made to the biosynthetic constructs in an effort to enhance production levels yet improved titers were not obtained.Additionally, this thesis will look at the development of a new strategy for the heterologous expression of biosynthetic pathways in a number of diverse hosts. I will highlight a flexible in vivo heterologous expression system that was inspired by viral protein packaging, processing and cleavage to produce violacein, a bright purple pigment with anti-tumor properties. A de novo polyprotein design possessing the violacein biosynthetic pathway was shown to work effectively in prokaryotic hosts such as E. coli and S. typhimurium. Expression of the polyprotein design in eukaryotic hosts like mammalian cells and S. cerevisiae were less successful. The ultimate goal of the work presented herein is to highlight the flexibility and powerful nature of synthetic biology for the in vivo production of natural products in addition to contributing to the vast arsenal of techniques and strategies that are currently available to researchers in this field.

(1) The field of synthetic biology is rapidly growing, empowered by advancements in Molecular Biology. To express genes of interest, scientists exploit plasmids engineered for bacterial or mammalian expression. Existing plasmids carry superfluous DNA that decreases transformation and transfection efficiencies. Here, we present a novel set of mammalian expression vectors with different selection markers and tunable expression levels. Despite being substantially smaller than traditional vectors, these minimized plasmids display similar levels of gene expression.This set of novel mammalian expression vectors should be useful for a broad range of modern applications. (2) DREAM is a mammalian calcium-dependent repressor of gene expression, which binds to a downstream DNA sequence, called DRE. The DREAM/DRE system offers a unique way of linking gene expression to calcium influx (44), but the utility of this system has not been assessed in bacteria. Here we develop a simple bacterial cell growth assay, where cell growth is prevented by the expression of SacB. This cell growth assay is then exploited to assess the utility of the DREAM/DRE system in bacteria. Unexpectedly, the DRE sequence by itself represses the expression of SacB, which can then be de-repressed by the co-expression of DREAM. Should this recovery of SacB expression maintain calcium-dependence, the DREAM/DRE system could be exploited in future directed strategies to evolve calcium-permeable ion channels.

I propose the use of the tandem fluorescent timer protein as a reporter of dynamic gene regulation. The tandem fluorescent timer is a fusion of two fluorophores with different maturation kinetics whose fluorescence ratio is a reporter of protein age. Traditional approaches to live single-cell tracking of dynamic gene expression involve the use of destabilized fluorescent reporters. The reduced stability of these reporters improve performance but also result in reduced signal and an increased signal to noise ratio. I first develop a platform to test reporter performance by designing and implementing an inducible synthetic network orthogonally in S. cerevisiae cells and by developing a microfluidics-enabled live cell-tracking pipeline. To test the performance of different reporters, I develop an algorithm to decode the underlying regulatory dynamic signal of a fluorescence profile. I then simulate the fluorescence output of my platform under dynamic regulatory signaling to demonstrate the potential reporter performance of a stable timer protein. Finally, I conduct live cell-tracking experiments of yeast cells expressing the timer under a periodic signal to test in vivo performance of the tandem fluorescent timer. I demonstrate that compared to a traditional stable fluorescent reporter, the tandem fluorescent timer is more accurate when tracking faster periodic signals and it is more robust to global fluctuations.

The next generations of both biological engineering and computer engineering demand that control be exerted at the molecular level. Creating, characterizing and controlling synthetic biological systems may provide us with the ability to build cells that are capable of a plethora of activities, from computation to synthesizing nanostructures. To develop these systems, we must have a set of tools not only for synthesizing systems, but also designing and simulating them. The BioJADE project provides a comprehensive, extensible design and simulation platform for synthetic biology. BioJADE is a graphical design tool built in Java, utilizing a database back end, and supports a range of simulations using an XML communication protocol. BioJADE currently supports a library of over 100 parts with which it can compile designs into actual DNA, and then generate synthesis instructions to build the physical parts. The BioJADE project contributes several tools to Synthetic Biology. BioJADE in itself is a powerful tool for synthetic biology designers. Additionally, we developed and now make use of a centralized BioBricks repository, which enables the sharing of BioBrick components between researchers, and vastly reduces the barriers to entry for aspiring Synthetic Biologists.

The study of synthetic gene regulatory networks allows the isolation and investigation of components and motifs in natural regulatory networks. Many synthetic gene networks are regulated at the transcriptional level. In this work, two methods of regulating gene expression at the transcriptional level were studied with the objective of gaining finer control over network behaviour. The first approach focuses on activation and repression of promoters by transcription factors. A synthetic repressor-activator network was engineered using the cI and cro genes and the PRM promoter from bacteriophage λ. The cI and cro genes activated and repressed PRM, respectively, and the monomeric red fluorescent protein (mrfp) gene reported PRM activity. Experimental testing showed an increase in mrfp expression in response to CI, a decrease in mrfp expression in response to Cro, and a differential output that reflected the relative concentrations of CI and Cro when both inputs were applied together. A positive feedback network was then created by placing a cI gene downstream of PRM. The network showed increased expression in response to CI and decreased expression in response to Cro. A negative feedback network was created by placing a cro gene downstream of PRM. Experimental testing showed decreased mrfp expression in response to both inputs. The second approach employed two methods for tuning expression levels without modifying the genes or promoters. First, using a series of networks with tandem mrfp genes under the control of the PLtet0-1 promoter, it was demonstrated that magnitude and range of expression levels could be tuned by adjusting the number of genes in the operon. A network was tuned using this principle by placing luxR genes in tandem to increase the activity of the luxPR promoter. It was then demonstrated that the level of gene expression could be varied through the placement of the gene within an operon. Operons that were three, five and seven genes and contained one green fluorescent protein gene in the first, middle, or end position were created. By comparing green fluorescence levels in induced and uninduced networks, it was found that the gene closest to the promoter was the most inducible.

The study of synthetic gene regulatory networks allows the isolation and investigation of components and motifs in natural regulatory networks. Many synthetic gene networks are regulated at the transcriptional level. In this work, two methods of regulating gene expression at the transcriptional level were studied with the objective of gaining finer control over network behaviour. The first approach focuses on activation and repression of promoters by transcription factors. A synthetic repressor-activator network was engineered using the cI and cro genes and the PRM promoter from bacteriophage λ. The cI and cro genes activated and repressed PRM, respectively, and the monomeric red fluorescent protein (mrfp) gene reported PRM activity. Experimental testing showed an increase in mrfp expression in response to CI, a decrease in mrfp expression in response to Cro, and a differential output that reflected the relative concentrations of CI and Cro when both inputs were applied together. A positive feedback network was then created by placing a cI gene downstream of PRM. The network showed increased expression in response to CI and decreased expression in response to Cro. A negative feedback network was created by placing a cro gene downstream of PRM. Experimental testing showed decreased mrfp expression in response to both inputs. The second approach employed two methods for tuning expression levels without modifying the genes or promoters. First, using a series of networks with tandem mrfp genes under the control of the PLtet0-1 promoter, it was demonstrated that magnitude and range of expression levels could be tuned by adjusting the number of genes in the operon. A network was tuned using this principle by placing luxR genes in tandem to increase the activity of the luxPR promoter. It was then demonstrated that the level of gene expression could be varied through the placement of the gene within an operon. Operons that were three, five and seven genes and contained one green fluorescent protein gene in the first, middle, or end position were created. By comparing green fluorescence levels in induced and uninduced networks, it was found that the gene closest to the promoter was the most inducible.