Synthetic Biology

Tools and Applications

Huimin Zhao

• Publisher: Academic Press
• Year: 2013
• Print ISBN: 9780123944306
• eBook ISBN: 9780123978202
• Topic: Biological Sciences
• Subtopic: Biotechnology
• Index: Biological Sciences

Synthetic Biology provides a framework to examine key enabling components in the emerging area of synthetic biology. Chapters contributed by leaders in the field address tools and methodologies developed for engineering biological systems at many levels, including molecular, pathway, network, whole cell, and multi-cell levels. The book highlights exciting practical applications of synthetic biology such as microbial production of biofuels and drugs, artificial cells, synthetic viruses, and artificial photosynthesis. The roles of computers and computational design are discussed, as well as future prospects in the field, including cell-free synthetic biology and engineering synthetic ecosystems.Synthetic biology is the design and construction of new biological entities, such as enzymes, genetic circuits, and cells, or the redesign of existing biological systems. It builds on the advances in molecular, cell, and systems biology and seeks to transform biology in the same way that synthesis transformed chemistry and integrated circuit design transformed computing. The element that distinguishes synthetic biology from traditional molecular and cellular biology is the focus on the design and construction of core components that can be modeled, understood, and tuned to meet specific performance criteria and the assembly of these smaller parts and devices into larger integrated systems that solve specific biotechnology problems.- Includes contributions from leaders in the field presents examples of ambitious synthetic biology efforts including creation of artificial cells from scratch, cell-free synthesis of chemicals, fuels, and proteins, engineering of artificial photosynthesis for biofuels production, and creation of unnatural living organisms- Describes the latest state-of-the-art tools developed for low-cost synthesis of ever-increasing sizes of DNA and efficient modification of proteins, pathways, and genomes- Highlights key technologies for analyzing biological systems at the genomic, proteomic, and metabolomic levels which are especially valuable in pathway, whole cell, and multi-cell applications- Details mathematical modeling tools and computational tools which can dramatically increase the speed of the design process as well as reduce the cost of development

Section III

Applications in Synthetic Biology

Chapter 9 Design and Application of Synthetic Biology Devices for Therapy

Chapter 10 Drug Discovery and Development via Synthetic Biology

Chapter 11 Synthetic Biology of Microbial Biofuel Production

Chapter 12 Tools for Genome Synthesis

Chapter 13 Synthetic Microbial Consortia and their Applications

Chapter 9

Design and Application of Synthetic Biology Devices for Therapy

Boon Chin Heng and Martin Fussenegger, ETH-Zurich, Basel, Switzerland

Introduction

Synthetic biology is the rational and systematic design/construction of biological systems with desired functionality.^1–4 This is achieved through applying engineering and computational principles within the field of molecular biology.^1–4 As such, synthetic biology represents an eclectic fusion of multiple disciplines. The debut of synthetic biology took place in the year 2000, when the first synthetic gene circuits, a toggle switch⁵ and an oscillator,⁶ were constructed in bacterial systems. Since then, the field has progressed extremely rapidly, with increasingly complex synthetic gene regulatory circuits being placed into mammalian cells,^7,8 fungi,^9,10 and viruses,^11,12 in addition to prokaryotic bacterial systems.^5,6,13 This, in turn, has spawned a diverse array of potential therapeutic applications, ranging from drug screening and discovery to cancer treatment and even fabrication of novel biomaterials.^14,15

In recent years, the pace of progress in synthetic biology has been further accelerated by the gradual adoption of a bioinformatics and computer-aided design approach to the construction of synthetic gene circuits.^16–20 A number of software tools have been specifically developed for synthetic biology applications. These include SynBioSS (Synthetic Biology Software Suite),²¹ TinkerCell,²² and BioNetCAD.²³ Such programs provide databases of the different potential building blocks of synthetic biological systems, and enable modeling and simulation of hypothetical gene circuits in silico before conducting actual experiments in vitro and in vivo.

This chapter will start by providing an overview of the target organisms and the molecular toolkit available for synthetic biology applications. This will be followed by an extensive review of the potential therapeutic applications of synthetic biology in various aspects of human health and disease. Finally, we will critically examine future challenges and safety issues associated with the application of synthetic biology to clinical practice.

Target Organisms and Cell Types for Therapeutic Applications of Synthetic Biology

Human cells and tissues appear to be the most direct targets for the engineering of therapeutic gene circuits,^14,24,25 particularly for cancer therapy, to correct aberrant metabolic conditions, and for application in regenerative medicine. However, tinkering with the biological functions of bacteria and viruses can also have useful clinical applications. The diverse population of commensal and symbiotic microorganisms associated with the human body, known as the human microbiome, forms a complex ecosystem that plays an important role in regulating human physiology, health, and disease states.^26,27 The fact that the human body naturally tolerates the various species of microorganisms associated with it makes these microorganisms ideal targets for synthetic biology applications. Indeed, synthetic gene circuits have been engineered in human microbiome-associated bacterial species to confer host resistance to infectious diseases such as cholera,²⁸ as well as to kill cancerous cells.²⁹ Besides bacterial cells, synthetic gene circuits can also be deployed in viruses for therapeutic applications. Of particular interest are the bacteriophages, which are viruses that specifically infect bacterial cells. The T7 phage has been engineered to express the bacterial biofilm-degrading enzyme dispersin B,³⁰ while a synthetic gene circuit that interferes with the SOS response network in bacterial cells has been engineered in the M13 phage.³¹

Molecular Toolkit for Synthetic Biology

Synthetic Gene Circuits Encoded by Recombinant DNA

Gene Targeting and Genome Editing Technologies

Transfection of recombinant DNA into mammalian or bacterial cells is an essential prerequisite for the deployment of synthetic gene circuits. The major challenge is to achieve efficient and site-specific integration of the transfected recombinant DNA into the host genome. Transient transfection with nonintegrating plasmid DNA imposes severe limitations on long-term clinical applications. Moreover, there is a low probability of random and nonsite-specific integration of the plasmid DNA into the host genome, which could cause insertional mutagenesis of host genes. This could potentially lead to cancer, and hence invoke serious safety concerns.³² Although the use of viral vectors can efficiently integrate recombinant DNA into the host genome, again, the problem is the random and nonsite-specific integration of recombinant DNA into the cellular host genome.^33,34

The development of site-specific recombinase (SSR) technology was the first attempt at site-specific manipulation of genomic DNA. SSR systems such as Cre-LoxP,³⁵ Flp-FRT,³⁶ Dre,³⁷ and PhiC31³⁸ can be used to delete, insert, or invert a segment of DNA flanked by specific recombination sites within genomic DNA, and have now been incorporated into viral vectors.^39,40 More recently, there have been advances in the rational design of zinc finger (ZF) nucleases for site-specific insertion of recombinant DNA.^41–44 The nonspecific Fok1 domain of ZF nucleases can be coupled to transcription activator-like effectors (TALEs) to form novel genome-editing tools that enable precise integration of recombinant DNA into target chromosomal locations.⁴⁵

It would also be of clinical interest to be able to transiently insert and remove transgenic elements in a precise and site-specific manner, without leaving any permanent modification to genomic DNA. For example, some synthetic biology applications may require temporary expression of synthetic gene circuits within cells for only a limited period of time. The PiggyBac transposon system can be particularly useful for this purpose.^46,47 Wilson et al.⁴⁸ demonstrated that PiggyBac integration and excision within human genomic DNA is very precise, without leaving any ‘footprint’ mutations at the site of transposon excision. The same study⁴⁸ also mapped a total of 575 PiggyBac integration sites within human genomic DNA to demonstrate the nonrandom site-selectivity of PiggyBac transposon integration. Such useful properties of the PiggyBac transposon system have been utilized for reprogramming mammalian somatic cells to pluripotent stem cells through the transient expression of four transgenes (c-Myc, Klf4, Oct4, and Sox2), without leaving any permanent genetic alteration to the reprogrammed cells.^49,50

To date, most synthetic gene networks have been constructed using the classical restriction–digestion-based molecular cloning method that has several inherent limitations. Multiple steps are often required, which makes this classical cloning technique both labor- and time-intensive. Moreover, the use of restriction enzymes would leave a restriction site-scar between annealed DNA fragments. Molecular cloning with specific restriction enzymes can also be potentially hindered by the presence of multiple restriction sites within either the target gene sequence or the destination vector backbone. These limitations may be overcome through newly developed restriction-enzyme free molecular cloning techniques that enable scarless, sequence-independent multipart DNA assembly, such as SLIC (sequence and ligase independent cloning),⁵¹ Gibson DNA assembly,⁵² and CPEC (circular polymerase extension cloning).⁵³ All that is required is for the target gene sequence to be PCR amplified with oligonucleotide primers that have 5′ termini sequence homology (at least 25 bp) to the corresponding ends of the destination vector. In the case of SLIC and Gibson DNA assembly, exonuclease activity is used to generate complementary overhangs on the target DNA fragment and the linearized destination vector, which are then annealed in the absence (SLIC) or presence (Gibson DNA assembly) of ligase. Another major difference between the two techniques is that T5 exonuclease is utilized in the case of Gibson DNA assembly, whereas SLIC utilizes T4 DNA polymerase which displays 3′ exonuclease activity in the absence of dNTPs.^51,52 By contrast, in the case of CPEC, no exonuclease activity is utilized to generate overhangs, and neither are oligonucleotide primers utilized.⁵³ Instead, both the target gene sequence and the linearized destination vector are melted into single strands and subsequently annealed to each other, thus allowing both DNA fragments to prime each other in the presence of Phusion polymerase.⁵³

Types of Synthetic Gene Circuits

Overview

The application of engineering and computing principles in synthetic biology has helped develop a diverse variety of synthetic gene circuits with a panoply of different functions. It is convenient to compare the functioning of synthetic gene circuits with analogous electronic devices. These are summarized in Table 9.1, and can be broadly classified into genetic switches,^5,54–67 oscillators,^6,54,68–72 filters,^73–79 communication modules,^80–83 and other miscellaneous synthetic gene circuits such as various digital logic gates.^84–89 Each of these is discussed here in turn.

Table 9.1

Different Types of Synthetic Gene Circuits

Genetic Switches

In electronics, a switch is a device that allows conditional transition from one state to another, in response to an input signal. Similarly, synthetic genetic switches are artificial regulatory networks that allow cells to undergo conditional transition between gene expression states. Although it may appear simple and straightforward to genetically engineer cells to switch on/off gene expression in response to metabolic, physical, or cytokine-induced stimuli, the challenge is to achieve a robust bistable transition from one state to another without the tendency to flip randomly between states as a result of fluctuations inherent in gene expression. This challenge may be overcome through the incorporation of positive and negative feedback loops.^5,54–57 Construction of bistable genetic ...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• Contributors
• Introduction
• Section I: Synthesis and Engineering Tools in Synthetic Biology
• Section II: Computational and Theoretical Tools in Synthetic Biology
• Section III: Applications in Synthetic Biology
• Section IV: Future Prospects
• Index