Abstract
Synthetic biology aims to engineer and redesign biological systems for useful real-world applications in biomanufacturing, biosensing and biotherapy following a typical design-build-test cycle. Inspired from computer science and electronics, synthetic gene circuits have been designed to exhibit control over the flow of information in biological systems. Two types are Boolean logic inspired TRUE or FALSE digital logic and graded analog computation. Key principles for gene circuit engineering include modularity, orthogonality, predictability and reliability. Initial circuits in the field were small and hampered by a lack of modular and orthogonal components, however in recent years the library of available parts has increased vastly. New tools for high throughput DNA assembly and characterization have been developed enabling rapid prototyping, systematic in situ characterization, as well as automated design and assembly of circuits. Recently implemented computing paradigms in circuit memory and distributed computing using cell consortia will also be discussed. Finally, we will examine existing challenges in building predictable large-scale circuits including modularity, context dependency and metabolic burden as well as tools and methods used to resolve them. These new trends and techniques have the potential to accelerate design of larger gene circuits and result in an increase in our basic understanding of circuit and host behaviour.
Highlights
In order to survive and reproduce, cells must sense a wide variety of inputs both external and internal
Further advances enabled use of linear DNA through protection from degradation by RecBCD through the addition of GamS protein (Sun et al 2014). Using this method a 4-piece genetic switch was assembled within 8 h (1 working day), using simple Golden Gate assembly and polymerase chain reaction (PCR) to create 4 linear sequences directly used for testing, in this case there was a lack of correlation between in vivo and in vitro results (Sun et al 2014)
The genetic sequences are deconstructed and reconstituted into an optimal sequence design to be used in the target host organism. These are ranked using important factors when implementing in vivo. Examples used in this case are toxicity effects on the host in terms of percentage growth reduction and dynamic range of the output for the circuit, measured in fold change between the ON and the OFF states the main elements of the system should be known and their interactions with the genetic and protein elements of the cell specified directly
Summary
In order to survive and reproduce, cells must sense a wide variety of inputs both external and internal. Both the input and the output must be able to be connected to and interact with upstream and downstream components and operate in the intended fashion (be modular), the signal output must be stable, exhibit low noise (random unintended fluctuations), and have a large ON:OFF ratio, or dynamic range (Bradley and Wang 2015). The 1-bit full adder was functionally constructed in mammalian consortia incorporating 22 separate gates distributed amongst 9 specialized cell types in a complex three-dimensional environment (Auslander et al 2017) Construction of such large scale genetic circuits are uncommon, large numbers of logic gates in single cells are scarce and require significant amounts of time and effort to work through an iteration of the design-build-test-learn cycle. Focus will be on the obstacles to modularity as well as context effects and metabolic burden
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