In this chapter, a set of basic genetic elements (i.e. the promoters and RBSs) have been quantitatively characterised in various biophysical and genetic contexts for the systematic forward engineering of synthetic circuits with the modular AND gate function. The initial implementation of the designed genetic AND gate provides us with an evidence to show that rationally designed biological systems may rarely work as might be initially intended. This is partly because the individual parts and modules are not sufficiently well characterised for purpose, and so the effective coupling together of them is not straightforward due to their different characteristics. Moreover, the behaviour of component modules characterised in one context may vary quite differently in another working condition (Klumpp et al., 2009; Tan et al., 2009) and configuration (Salis et al., 2009). By quantitatively characterising a set of promoters, a series of RBSs and the assembled circuits in various contexts, both the biophysical and genetic contexts, in which they behave, were found to have a great impact on their functionalities. Experimental results in this chapter show that the variations of the behaviour of genetic parts and devices in different contexts are due to the many factors affecting gene expression, such as the cell chassis background, medium, temperature, the embedded sequence context. Thus the thorough characterisation of them in various conditions and configurations, particularly in the context of interest, are necessary for any successful wide reuse in the community and more importantly for facilitating the functional assembly of individual parts and modules into customisable large scale systems. While the creation of diverse part and module variants are becoming simplified and at reduced cost (Dougherty and Arnold, 2009; Ellis et al., 2009), high throughput and accurate technology platforms need to be established to accelerate the process of characterisation, like the recently established BIOFAB (biofab, 2010) open technology platform.
The modular genetic AND gate engineered here works as a fundamental module for regulating genetic information transmission in living cells, which can integrate two input signals to generate one output in the digital logic AND manner. Moreover, the core elements of this device are from the specialised hrp gene regulatory system
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for Type III secretion in Pseudomonas syringae, a plant pathogenic bacterium genetically far away from the standard E. coli chassis. Thus, the novel genetic AND gate module is likely orthogonal to the E. coli genetic background and can be used simultaneously with existing gene regulatory elements that are widely utilised for regulating gene expression in E. coli without any compromise. The orthogonality of the AND gate has been demonstrated by the experimental study in this chapter. The study also provides an example to exploit the diverse natural biological modules and to engineer them for creating novel orthogonal parts and modules to expand the limited toolbox of synthetic biology at the current stage. With the increase of the number orthogonal parts and modules in the toolbox, we can engineer more complicated systems which can contain many parts to enable the cells with high level functions.
From the fitted transfer function of the AND gate (Figure 4.15), it was noticed that the fit Hill constants and coefficients for the two activators HrpR and HrpS are quite different. This indicates that the two enhancer binding proteins likely have unequal roles in the binding and activation of the σ54-dependent hrpL promoter although they are originally expressed from the same operon and have many sequence homologies. On this aspect, the work corroborates that the study of simple synthetic biological circuits can contribute to the uncovering of the design principles of their natural counterparts (Mukherji and van Oudenaarden, 2009). In addition, the characterisation scope for synthetic parts and devices was extended. The AND gate circuit was subjected to homogeneity, metabolic load and chassis compatibility assays respectively beyond the normal population-level phenotype assays. The thorough characterisation of parts and devices will enable their wide reuse in the community and facilitate the reliable prediction of their behaviour when integrated into other larger systems.
The work in this chapter also shows that the functional assembly of component parts into devices and systems can be executed more predictably and reliably using engineered, in-context quantitatively characterised parts and sub-modules aided by modelling. This approach minimises the unexpected or high-order effects which could
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occur during circuit construction by characterising components in the same biophysical and genetic context as anticipated in their target system. This was illustrated by converting an assembled non-functional logic AND circuit to be a functional one. The modular AND gate can be reconnected to different sensor inputs to detect and integrate various environmental signals (Anderson et al., 2007; Kobayashi et al., 2004; Voigt, 2006) or easily incorporated into a large system as a fundamental building block to regulate the cell signalling in a desired logic manner. The behaviour of the constructed modules are well captured by the parameterised mathematical models for their transfer functions, which are reusable for modelling the behaviour of larger integrated modules (Guido et al., 2006). The functional assembly approach described here provides an effective strategy and a guide for the engineering of synthetic gene circuits with predictable functions across different contexts for their application in many areas such as biotechnology, biocomputing and biosensors.
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