Using two-component systems and other bacterial regulatory factors for the fabrication of synthetic genetic devices

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.

Figure 1. Basic circuit topology of the synthetic genetic clock

The activator module (left) consists of a promoter, Pa, that drives the expression of activator, A. The repressor module (right) consists of a promoter, Pr, that drives the expression of repressor, R. Repressor block transcription from Pa.

Figure 2. Structure of the synthetic genetic clock

The activator module (left) consists of a modified version of the glnA promoter, driving the expression of glnG (ntrC). Light blue boxes in the glnA control region signify the glnAp2 enhancer, dark blue boxes signify “perfect” lac operator sites, and grey boxes signify the glnAp2 governor sites. The relative positions of the glnAp1 and glnAp2 transcription start sites are shown by bent arrows. The product of glnG (ntrC), NRI, is converted to its active, phosphorylated, form by NRII2302, which is provided in excess from a plasmid (not depicted). NRI~P increases its own expression by binding to the enhancer, wherupon it represses glnAp1 and activates glnAp2. The repressor module (right) consists of the natural glnK promoter and translation initiation region fused to the lacI structural gene. The glnK promoter is associated with a weak enhancer (signified by a light blue box and a stippled light blue box). The product of the repressor module, LacI, blocks transcription from the activator module, and from the native lacZYA operon (bottom). Repression of the lacZYA operon is due to repressor interaction with the three operators of the this operon (dark blue boxes), as indicated. The product of lacZ, β-galactosidase, serves as a reporter for oscilatory behavior.

Figure 3. The activator module is an N-IMPLIES gate with positive feedback

When in cells containing wild-type natural components of the Ntr system except for NRI, containing the activator module to provide wild-type NRI, containing the wild-type lacI gene, and containing a fusion of lacZ to the glnK promoter, the activator module provides an N-IMPLIES logic gate for the regulation of β-galactosidase by ammonia and IPTG.

Figure 4. The activator module can function as a toggle switch

The basic circuit topology for the toggle switch is shown at top. The activator module produces activator that drives expression of GS and the activator module. LacI is produced from the natural chromosomal lacI gene, and represses the activator module as well as the natural lacZYA (not depicted). At bottom, typical results for lacZ expression (left), and glutamine synthetase expression (right) are shown for induced (dot) and naive (+) cultures. The overlapping curves for the lacZ expression data show that the steady state was reached; the GS data shows hysteresis of the activator module. An intuitive explaination for this hysteresis is as follows: When the level of NRI~P is high, as in the induced culture, it is difficult for repressor to get control of the system, and thus repression is only achieved when the IPTG concentration is low. In the naive cell, the level of NRI~P is very low and thus it is considerably easier for repressor to get control of the system. Thus, in naive cells, the system stays repressed even at fairly high concentrations of IPTG.

Figure 5. The repressor module functions as an OR logic gate

When the repressor module is present in cells containing a mutation in the natural lacI gene, and with otherwise wild-type Ntr system and lacZYA, it provides OR gate function with regard to ammonia and IPTG for expression of lacZYA.

Figure 6. Results of clock experiments using a standard laboratory continuous culture device

The activator module used in the experiments is depicted at top left. The experiments were conducted as described in text, with manual control of the nutrient pump to maintain stable culture turbidity. At top right, results are shown from an experiment where OD₆₀₀ and β-galactosidase were monitored. At bottom left, another experiment where glutamine synthetase was also measured is shown, for clarity the OD₆₀₀ data is not shown for this experiment. As predicted by the model, the expression of glutamine synthetase and β-galactosidase was out of phase. At bottom right, the phase diagram for the β-galactosidase and glutamine synthetase expression data is shown. Note that if the system contained a perfect oscillator, the phase diagram would reveal a stable orbit instead of spiraling inward to a steady state.

Figure 7. Schematic diagram for a home-made turbidostat

For details and further description, see text.

Figure 8. Close-up view of the reactor for a home-made turbidostat

For explaination, see text.

Figure 9

Results of a clock experiment employing an automated system and miniaturized β-galactosidase assay.

Figure 10. The pStep0 plasmids that can be used to create new landing pads

The vector backbone consists of the EcoRI-SapI fragment of pBR322 containing the origin of replication (triangle) and ampicillin resistance gene (AB0). The plasmids contain a linker with multiple unique sites in either of two sequences, denoted “t” and “c”.

Figure 11. The pStep1-pStep4 plasmids, that can be used to place genetic modules onto the E. coli chromosome

For the pStep1 plasmids, T signifies the target for recombination with the chromosome, P1 signifies a module promoter, and lollipop symbols signify transcriptional terminators. The selective marker for pStep1 recombination is AB1. For the pStep2 plasmids, AB1 fragments constitute the target for recombination with the chromosome, P2 signifies a module promoter, and the selective marker for recombination is AB2. For the pStep3 plasmids, AB2 fragments constitute the target for recombination with the chromosome, P3 signifies a module promoter, and the selective marker for recombination is AB1. For the pStep4 plasmids, AB1 fragments constitute the target for recombination with the chromosome, P4 signifies a module promoter, and AB2 provides the selective marker for recombination. Note that the “t” and “c” series of plasmids differ in the arrangement of internal sites of the linker. For all plasmids, AB0 is the ampicillin resistance gene, containing the PstI site that may be used for plasmid linearization.

Figure 12. The sequence of nesting that occurs upon succesive use of the pStep1-pStep4 plasmids for integration of modules into the chromosome

Symbols are as in Fig 11; G1-G4 depict structural genes within the modules, expressed from module promoters P1-P4. Recursive use of the pStep1-pStep4 plasmids to modify a chromosomal site results in side-by-side integration of modules on the chromosome, separated by transcriptional terminators.