AssemblyTron: flexible automation of DNA assembly with Opentrons OT-2 lab robots

Abstract

As one of the newest fields of engineering, synthetic biology relies upon a trial-and-error Design-Build-Test-Learn approach to simultaneously learn how function is encoded in biology and attempt to engineer it. Many software and hardware platforms have been developed to automate, optimize, and algorithmically perform each step of the Design-Build-Test-Learn cycle. However, there are many fewer options for automating the Build step. Build typically involves DNA assembly, which remains manual, low throughput, and unreliable in most cases and limits our ability to advance the science and engineering of biology. Here, we present AssemblyTron: an open-source python package to integrate j5 DNA assembly design software outputs with build implementation in Opentrons liquid handling robotics with minimal human intervention. We demonstrate the versatility of AssemblyTron through several scarless, multipart DNA assemblies beginning from fragment amplification. We show that AssemblyTron can perform PCRs across a range of fragment lengths and annealing temperatures by using an optimal annealing temperature gradient calculation algorithm. We then demonstrate that AssemblyTron can perform Golden Gate and homology-dependent in vivo assemblies with comparable fidelity to manual assemblies by simultaneously building four four-fragment assemblies of chromoprotein reporter expression plasmids. Finally, we used AssemblyTron to perform site-directed mutagenesis reactions via homology-dependent in vivo assembly also achieving comparable fidelity to manual assemblies as assessed by sequencing. AssemblyTron can reduce the time, training, costs, and wastes associated with synthetic biology, which along with open-source and affordable automation, will further foster the accessibility of synthetic biology and accelerate biological research and engineering.

Automated in vivo enzyme engineering accelerates biocatalyst optimization

“…Emerging biofoundries around the globe are providing automation capabilities for setting up such workflows 19,20,150 by transferring and combining available methods for modular DNA assembly, highly parallel transformation and incubation, imagebased colony identification and multi-pin picking, as well as plasmid library preparation using canonical hosts such as E. coli 151,152 or S. cerevisiae 153 as a basis. Recently, robotics-assisted modular cloning [154][155][156][157][158] , high-throughput transformation 159 and monoclonal colony cultivation and picking 160 have been introduced for other industrially relevant organisms such as P. putida or Corynebacterium glutamicum.…”

Biofoundry-assisted Golden Gate cloning with AssemblyTron

“…AssemblyTron is an open-source python package that, when paired with an Opentrons OT-2 lab robot, can provide biofoundry capabilities with minimal startup investment ( 16 ). AssemblyTron 1.0 uses j5 DNA design files as input and generates fully customized OpenTrons Application Program Interface (API) protocol scripts for assembling plasmids via homology mediated assembly or Golden Gate.…”

“…AssemblyTron 1.0 uses j5 DNA design files as input and generates fully customized OpenTrons Application Program Interface (API) protocol scripts for assembling plasmids via homology mediated assembly or Golden Gate. We previously validated that AssemblyTron is adept at automating multi-part Golden Gate assembly and can simultaneously increase throughput, eliminate human errors, and reduce training demands ( 16 ). AssemblyTron adds to a very sparse field of open-source build tools for automating Golden Gate assembly.…”

TidyTron: Reducing lab waste using validated wash-and-reuse protocols for common plasticware in Opentrons OT-2 lab robots

“…Generating a wash-and-reuse protocol library for the OT-2 would enable labs to mitigate single-use plastic waste without sacrificing time and energy of lab personnel. Many labs are already equipped with OT-2 liquid handling robots that automate more central protocols such as next generation sequencing library preparation 14 , diagnostic testing 15 , DNA assembly 16 , etc. The Opentrons OT-2 liquid handling robot equipped with a multichannel pipette is available for $11,000 17 .…”