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

Every year biotechnology labs generate a combined total of ∼5.5 million tons of plastic waste. As the global bioeconomy expands, biofoundries will inevitably increase plastic consumption in-step with synthetic biology scaling. Decontamination and reuse of single-use plastics could increase sustainability and reduce recurring costs of biological research. However, throughput and variable cleaning quality make manual decontamination impractical in most instances. Automating single-use plastic cleaning with liquid handling robots makes decontamination more practical by offering higher throughput and consistent cleaning quality. However, open-source, validated protocols using low-cost lab robotics for effective decontamination of plasticware-facilitating safe reuse-have not yet been developed. Here we introduce and validate TidyTron: a library of protocols for cleaning micropipette tips and microtiter plates that are contaminated with DNA, E. coli, and S. cerevisiae. We tested a variety of cleaning solutions, contact times, and agitation methods with the aim of minimizing time and cost, while maximizing cleaning stringency and sustainability. We tested and validated these cleaning procedures by comparing fresh (first-time usage) versus cleaned tips and plates for contamination with cells, DNA, or cleaning solutions. We assessed contamination by measuring colony forming units by plating, PCR efficiency and DNA concentration by qPCR, and event counts and debris by flow cytometry. Open source cleaning protocols are available at https://github.com/PlantSynBioLab/TidyTron and hosted on a graphical user interface at https://jbryantvt.github.io/TidyTron/.

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

As one of the newest fields of engineering, synthetic biology relies upon a trial-and-error Design-Build-Test-Learn (DBTL) approach to simultaneously learn how a 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 DBTL cycle. However, there are many fewer options for automating the build step. Build typically involves deoxyribonucleic acid (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 polymerase chain reactions 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 (IVAs) 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 IVA 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.