End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

Abstract

Background: Synthetic biology aims to engineer biological systems for desired behaviors. The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening.

Results: Herein, we present a programmable, multipurpose microfluidic platform and associated software and apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis. We show the platform's capabilities for multiple automated DNA assembly methods, including a new method for Isothermal Hierarchical DNA Construction, and for Escherichia coli and Saccharomyces cerevisiae transformation. The platform enables the automated control of cellular growth, gene expression induction, and proteogenic and metabolic output analysis.

Conclusions: Taken together, we demonstrate the microfluidic platform's potential to provide end-to-end solutions for synthetic biology research, from design to functional analysis.

Keywords: Analysis; Cell culture; DNA assembly; Microfluidics; Synthetic biology; Transformation.

Fig. 1

Synthetic biology research cycle for the development of new biological systems

Fig. 2

Microfluidics chip. a Photograph of the physical microfluidics chip (ruler for scale). 16 input/output macro scale wells (~20 uL working volume; blue-colored regions) surround internal micro scale valves (~150 nL). b PR-PR software user-interface schematic representation of the microfluidics chip. The red arrows show a representative example of a reagent transfer path through the chip from macro scale input well [21] to macro scale output well [8], through internal micro scale values [1, 5, 9, 13, 17, 20]

Fig. 3

Isothermal hierarchical DNA construction (IHDC) coupled with Gibson DNA assembly. a Isothermal hierarchical DNA construction. At left, an example of a three-level hierarchical construction tree starting with eight oligonucleotides. At right, schematic description of the basic biochemical step of IHDC. b Gibson DNA assembly. Integration of IHDC-constructed fragments with Gibson DNA assembly

Fig. 4

Overview of isothermal DNA construction on the microfluidics platform. a Overview of the basic IHDC step on the microfluidics platform. Stage I. Two oligos A and B (as shown, or alternatively two DNA fragments A and B) and a mixture of enzymes are transferred to the reactor. Stage II. Primers P1 and P2, and a mixture of enzymes, are transferred to the reactor. Stage III. A mixture of ATP and magnesium acetate, and a mixture of enzymes, are transferred to the reactor. The temperature is increased to 38 °C, and the reaction is incubated for 15 min. As a result, an elongated and amplified DNA fragment AB is produced. b Hierarchical construction tree of seven separate synthesis reactions that result in the final product (gfp as shown). c Gel electrophoresis image of all the intermediates and the final gfp construct. Lanes as labelled by: M: GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific); Level 1 (quarter) fragments: 1-2, 3-4, 5-6, 7-8; Level 2 (half) fragments: 1-4 and 5-8; Level 3 (full length gfp) fragment: 1-8

Fig. 5

Construction in yeast of a promoter library. a Promoter library schematic. b Gel electrophoresis image of amplified promoters: lane 1, Gal-250; lane 2, Gal-100; lane 3, Leu-250; lane 4, spo-250; lane 5, spo-100; lane 6, tef-250; lane 7, tef-100. c Gel electrophoresis image of: lane 1, pRS426; lane 2, pRS426-yeGFP EagI digest. M is GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific). d Schematic of reagent transfers through the microfluidic chip. The green input wells contain a mixture of different promoters and digested plasmid pRS426 with Salomon sperm DNA as a carrier. The yellow input wells contain S. cerevisiae competent cells. Arrows show pathways of reagent transfer on-chip according to the automated protocol

Fig. 6

Representation of the microfluidics platform’s functional modules. The platform is built from microfluidic and computational modules