Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly

Abstract

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly. Importantly, our designed UNSes make it possible to assemble parts with repeated terminator and insulator sequences, and thereby create insulated functional genetic circuits in bacteria and mammalian cells. Using UNS-guided assembly to construct repeating promoter-gene-terminator parts, we systematically varied gene expression to optimize production of a deoxychromoviridans biosynthetic pathway in Escherichia coli. We then used this system to construct complex eukaryotic AND-logic gates for genomic integration into embryonic stem cells. Construction was performed by using a standardized series of UNS-bearing BioBrick-compatible vectors, which enable modular assembly and facilitate reuse of individual parts. UNS-guided isothermal assembly is broadly applicable to the construction and optimization of genetic circuits and particularly those requiring tight insulation, such as complex biosynthetic pathways, sensors, counters and logic gates.

Bricks and blueprints: methods and standards for DNA assembly

“…Software tools will increasingly play an important role in DNA assembly, and are already required for the design of parts for modular DNA assembly 54,55,59 and used in the experimental planning and quality control of large and complex projects [67][68][69] . Many of these software tools can also control liquid handling robots and their ability to automate hundreds of complex modular assemblies has been recently demonstrated 62 .…”

“…An alternative approach avoids PCR by instead adding the linkers through cloning the parts into a plasmid where the MCS is already flanked by the appropriate linker sequences. The linker-flanked parts can then be excised using restriction endonucleases that cut outside the linkers, and the linear DNA can then be assembled by the Gibson method 55 .…”

“…By formatting parts and protocols according to a standard, future workflows will be set so that parts can be combined efficiently over sequential tiers and exchanged between projects around the world. As these standards are implemented the next logical steps for DNA assembly should recapitulate mature engineering disciplines: enabling software will be developed, automation of the labour will be introduced and ultimately the work of cloning will be commercially outsourced just as it is already done for gene synthesis and DNA sequencing.Software tools will increasingly play an important role in DNA assembly, and are already required for the design of parts for modular DNA assembly 54,55,59 and used in the experimental planning and quality control of large and complex projects [67][68][69] . Many of these software tools can also control liquid handling robots and their ability to automate hundreds of complex modular assemblies has been recently demonstrated 62 .…”

Synthetic Botany

“…Recently, several alternative DNA assembly technologies have gained prominence because they offer the advantage of allowing the assembly of multiple parts in a single reaction (Engler et al 2008;Gibson et al 2009;Quan and Tian 2009;Li and Elledge 2012;De Kok et al 2014). Although overlap-dependent methods such as Gibson assembly are powerful, they require custom oligonucleotides and amplification of even well characterized standard parts for each new assembly (Ellis et al 2009;Liu et al 2013;Patron 2014) unless standardized overlaps are used (Torella et al 2013;Casini et al 2014). The application of Type IIS restriction enzymes for assembling standard parts, known widely as "Golden Gate Cloning," has become widely used as an alternative approach because parts can be exchanged and assembled cheaply, easily, and in an automatable way without proprietary tools and reagents (Engler et al 2009;Sarrion-Perdigones et al 2011;Werner et al 2012).…”

Efficient Search, Mapping, and Optimization of Multi-protein Genetic Systems in Diverse Bacteria

“…Combinatorial assembly methods enable the simultaneous introduction and modification of genetic parts to create many genetic system variants (Engler et al, 2009;Gibson et al, 2009;Guye et al, 2013;de Raad et al, 2013;Sleight & Sauro, 2013;Coussement et al, 2014;Dharmadi et al, 2014;Torella et al, 2014). The development of multiplex genome engineering provides the ability to simultaneously introduce DNA mutations into several genomic loci (Wang et al, 2009(Wang et al, , 2012Esvelt & Wang, 2013).…”