DNA-BOT: a low-cost, automated DNA assembly platform for synthetic biology

Abstract

Multi-part DNA assembly is the physical starting point for many projects in Synthetic and Molecular Biology. The ability to explore a genetic design space by building extensive libraries of DNA constructs is essential for creating programmed biological systems. With multiple DNA assembly methods and standards adopted in the Synthetic Biology community, automation of the DNA assembly process is now receiving serious attention. Automation will enable larger builds using less researcher time, while increasing the accessible design space. However, these benefits currently incur high costs for both equipment and consumables. Here, we address this limitation by introducing low-cost DNA assembly with BASIC on OpenTrons (DNA-BOT). For this purpose, we developed an open-source software package and demonstrated the performance of DNA-BOT by simultaneously assembling 88 constructs composed of 10 genetic parts, evaluating the promoter, ribosome binding site and gene order design space for a three-gene operon. All 88 constructs were assembled with high accuracy, at a consumables cost of $1.50-$5.50 per construct. This illustrates the efficiency, accuracy and affordability of DNA-BOT, making it accessible for most labs and democratizing automated DNA assembly.

Keywords: DNA assembly; automation; biofoundry; synthetic biology.

Figure 1.

BASIC DNA assembly. (a) BASIC provides an assembly format where each DNA ‘part’ is joined with a linker. The parts of interest are assembled into a standard backbone, comprised of an origin of replication (ORI) and an antibiotic resistance marker (A^R). The assembled parts are flanked by linkers that recapitulate the BASIC Prefix and Suffix (LMP and LMS), thus generating the construct in the idempotent BASIC standard. (b) DNA parts are usually stored in high-copy number ampicillin resistance vectors, flanked by the BASIC Prefix and Suffix sequences with BsaI restriction sites used to release the parts from the vectors. (c) Linkers are synthetic oligonucleotides annealed to form half-linkers with 4 bp overhangs that are specific for the Prefix and Suffix overhangs and 21 bp single-stranded overhangs that direct the assembly; each half-linker is ligated to specific parts in separate clip reactions. Linkers used in this study are either UTR-RBS linkers that encode an RBS within a defined 5′-UTR; or methylated Prefix and Suffix linkers; fusion linkers can also be used to create fusion proteins and neutral linkers are available with no defined function (4).

Figure 2.

BASIC DNA assembly and the DNA-BOT workflow. (a) BASIC DNA assembly workflow: Step 1: Clip reaction: simultaneous digestion and ligation attaches Prefix (P-) and Suffix (S-) half-linkers to parts. Step 2: Purification: clips are purified from the reactions via solid phase reversible immobilization (SPRI), removing excess linkers and enzymes. Step 3: Assembly: purified clips are annealed, forming circular constructs e.g. part A and B are annealed with a backbone (bb) part in a three-part assembly. Step 4: Transformation: assembled constructs are transformed into E. coli. (b) csv files describing source plates for parts, linkers and construct designs are processed by the DNA-BOT application (app), returning four OT-2 scripts along with meta-information files. Each script runs the corresponding BASIC step in microtiter plate format, finally spotting colonies on selective LB-agar plates. Dotted lines denote information flow and solid-lines denote physical processes.

Figure 3.

DNA-BOT provides for efficient and accurate DNA assembly. Relative promoter and RBS strengths are indicated by gradients. (a) Design of the 88 constructs assembled using DNA-BOT. The library contained full permutations of promoters and RBSs as indicated in two gene orders for the sfGFP (GFP), mCherry (RFP) and mTagBFP (BFP) genes, with the exception of the weak J23105 promoter and RBS1 GFP combination. (b) Image of the agar plate on a Safe Imager™ 2.0 Blue Light Transilluminator, acquired following the DNA-BOT workflow with 10 µl of each transformation reaction spotted. Operon design features for each well position are indicated at axes of panels b and c. Green colonies indicate strong sfGFP expression. (c) Green, orange and blue bars denote normalized mean superfolder GFP (GFP), mCherry (RFP) and mTagBFP (BFP) fluorescence, respectively, measured via flow cytometry for each construct from three biological repeats. Fluorescence is log scaled. Error bars denote standard deviations between three biological repeats.