Registry in a tube: multiplexed pools of retrievable parts for genetic design space exploration

Abstract

Genetic designs can consist of dozens of genes and hundreds of genetic parts. After evaluating a design, it is desirable to implement changes without the cost and burden of starting the construction process from scratch. Here, we report a two-step process where a large design space is divided into deep pools of composite parts, from which individuals are retrieved and assembled to build a final construct. The pools are built via multiplexed assembly and sequenced using next-generation sequencing. Each pool consists of ∼20 Mb of up to 5000 unique and sequence-verified composite parts that are barcoded for retrieval by PCR. This approach is applied to a 16-gene nitrogen fixation pathway, which is broken into pools containing a total of 55 848 composite parts (71.0 Mb). The pools encompass an enormous design space (1043 possible 23 kb constructs), from which an algorithm-guided 192-member 4.5 Mb library is built. Next, all 1030 possible genetic circuits based on 10 repressors (NOR/NOT gates) are encoded in pools where each repressor is fused to all permutations of input promoters. These demonstrate that multiplexing can be applied to encompass entire design spaces from which individuals can be accessed and evaluated.

MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

“…Thirdly, an assembly vector of type ‘End’ generates assembled fragments that start with adhesive end ‘a’, ‘b’, ‘c’, ‘d’ or ‘e’, and end with adhesive end ‘q’ (Figure 4 ). The adhesive ends designed for orderly multi-fragment DNA assembly were chosen based on a previous design to minimize cross-reactivity between non-compatible adhesive ends ( 25 ). We have designed the adhesive ends ‘p’ and ‘q’ such that the vector set is compatible with several existing part libraries for mammalian and plant synthetic biology ( 10 , 12 , 26 ), as well as the OpenPlant synthetic biology standard ( 27 ).…”

Automated Robotic Liquid Handling Assembly of Modular DNA Devices

“…Each transcriptional unit is a linear arrangement of promoter, ribosomal binding site, gene, and transcriptional terminator, cloned into a specific destination vector. TUs are a key component in many hierarchical genetic circuit designs 28 29 30 and are therefore a natural proof of concept for this approach. Sequence-verified clones can be achieved in ~5 days from start to finish, and an overview of the presented workflow is presented in Figure 1a .…”

Engineering biological systems using automated biofoundries

“…Given the ever-increasing resolution, scanning speed, and quantification capability in MS analyzers, direct infusion methods may be used for higher throughput. For example, Agilent RapidFire platform couples solid-phase extraction with HT-ESI-MS for rapid and automated MS analysis (Woodruff et al, 2016). Moreover, MALDI mass spectrometry imaging has also been increasingly used for high-throughput screening thanks to its rapid speed, high tolerance to salts, and simple sample preparation (Zhou et al, 2015).…”