Elsevier

Methods in Enzymology

Volume 422, 2007, Pages 488-512
Methods in Enzymology

[25] - Using Two‐Component Systems and Other Bacterial Regulatory Factors for the Fabrication of Synthetic Genetic Devices

https://doi.org/10.1016/S0076-6879(06)22025-1Get rights and content

Abstract

Synthetic biology is an emerging field in which the procedures and methods of engineering are extended living organisms, with the long‐term goal of producing novel cell types that aid human society. For example, engineered cell types may sense a particular environment and express gene products that serve as an indicator of that environment or affect a change in that environment. While we are still some way from producing cells with significant practical applications, the immediate goals of synthetic biology are to develop a quantitative understanding of genetic circuitry and its interactions with the environment and to develop modular genetic circuitry derived from standard, interoperable parts that can be introduced into cells and result in some desired input/output function. Using an engineering approach, the input/output function of each modular element is characterized independently, providing a toolkit of elements that can be linked in different ways to provide various circuit topologies. The principle of modularity, yet largely unproven for biological systems, suggests that modules will function appropriately based on their design characteristics when combined into larger synthetic genetic devices. This modularity concept is similar to that used to develop large computer programs, where independent software modules can be independently developed and later combined into the final program. This chapter begins by pointing out the potential usefulness of two‐component signal transduction systems for synthetic biology applications and describes our use of the Escherichia coli NRI/NRII (NtrC/NtrB) two‐component system for the construction of a synthetic genetic oscillator and toggle switch for E. coli. Procedures for conducting measurements of oscillatory behavior and toggle switch behavior of these synthetic genetic devices are described. It then presents a brief overview of device fabrication strategy and tactics and presents a useful vector system for the construction of synthetic genetic modules and positioning these modules onto the bacterial chromosome in defined locations.

Section snippets

Using Two‐Component Signal Transduction Systems in Synthetic Biology Approaches

Two‐component signaling systems have been studied intensively since the mid‐1980s and provide numerous examples of systems where the cellular physiology of the regulatory phenomena and the activities of the signal transduction components are reasonably well understood (Hoch and Silhavy, 1995). Certain aspects of these signal transduction systems make them particularly useful for synthetic biology purposes. Foremost among these is that many two‐component systems are not essential for viability

Using the NRI/NRII System to Build a Synthetic Genetic Clock

The basic circuit topology for the synthetic genetic clock is shown in Fig. 1. The clock consists of two modules: activator and repressor. The activator module (Fig. 1, left) consists of a promoter that drives the expression of the activator, and is itself activated by the activator. The activator also drives the expression of the repressor module (Fig. 1, right), which produces the repressor. The repressor protein blocks the expression of the activator module. Modeling of this circuit

Fabrication of Synthetic Genetic Clock

A unique aspect of our synthetic genetic clock is that the activator and repressor modules are not contained on plasmids in the cell, but rather are integrated into defined chromosomal locations, referred to as “landing pads” (Atkinson et al., 2003). We expect that this should provide a stable copy number to the modules, and indeed allows subtle manipulation of the copy number by using different locations on the chromosome, as the copy number of genes in rapidly growing cells displays a

Functions of Individual Clock Modules

Activator module and repressor module functions can be measured independently in intact cells. The activator module, when present in cells containing wild‐type NRII and wild‐type lacI encoding the Lac repressor, is predicted to form an N‐IMPLIES logic gate with respect to ammonia and IPTG (Fig. 3). In the presence of wild‐type NRII, the nitrogen‐rich state brought about by the presence of ammonia causes formation of the NRII–PII complex and rapid dephosphorylation of NRI∼P. Furthermore, in the

Improved Procedures for Fabrication of Synthetic Genetic Modules and Integration of These Modules into Chromosomal Landing Pads

During our synthetic biology studies, we have developed fabrication methods in concert with development of the clock, such that early versions of the clock do not incorporate the most useful aspects of fabrication methodologies developed later. This chapter presents our most recent vector system for the fabrication of synthetic genetic modules and incorporation of the fabricated modules into chromosomal landing pads. The vector system should allow placement of any module in any position and on

Fabricating Genetic Modules

Most of the genetic modules that we fabricate consist of a regulated promoter that drives the expression of a regulatory gene. The minimal components therefore consist of a bacterial promoter, regulatory sites for control of the promoter, an mRNA leader sequence containing translational initiation sequences, the structural gene for the desired regulatory protein, and a transcriptional terminator sequence. For genetic isolation, it is advisable to also include transcriptional termination

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