basicsynbio and the BASIC SEVA collection: software and vectors for an established DNA assembly method

Abstract

Standardized DNA assembly methods utilizing modular components provide a powerful framework to explore designs and iterate through Design-Build-Test-Learn cycles. Biopart Assembly Standard for Idempotent Cloning (BASIC) DNA assembly uses modular parts and linkers, is highly accurate, easy to automate, free for academic and commercial use and enables simple hierarchical assemblies through an idempotent format. These features enable applications including pathway engineering, ribosome binding site tuning, fusion protein engineering and multiplexed gRNA expression. In this work we present basicsynbio, open-source software encompassing a Web App (https://basicsynbio.web.app/) and Python Package (https://github.com/LondonBiofoundry/basicsynbio), enabling BASIC construct design via simple drag-and-drop operations or programmatically. With basicsynbio, users can access commonly used BASIC parts and linkers while designing new parts and assemblies with exception handling for common errors. Users can export sequence data and create instructions for manual or acoustic liquid-handling platforms. Build instruction generation relies on the BasicBuild Open Standard which is parsed for bespoke workflows and is serializable in Java Script Object Notation for transfer and storage. We demonstrate basicsynbio, assembling 30 vectors using sequences including modules from the Standard European Vector Architecture (SEVA). The BASIC SEVA vector collection is compatible with BASIC and Golden Gate using BsaI. Vectors contain one of six antibiotic resistance markers and five origins of replication from different compatibility groups. The collection is available via Addgene under an OpenMTA agreement. Furthermore, vector sequences are available from within the basicsynbio application programming interface with other collections of parts and linkers, providing a powerful environment for designing assemblies for bioengineering applications.

Revealing the Host-Dependent Nature of an Engineered Genetic Inverter in Concordance with Physiology

“…We therefore built a tractable experimental platform by transforming six Gammaproteobacteria species with a genetic inverter circuit cloned onto a pSEVA231-derived (kanamycin selection marker and pBBR1 origin of replication) vector (Fig. 1 A and B) within the BASIC cloning environment [ 29 , 30 ], yielding plasmid pS4. The inverter consists of 2 inducible antagonistic expression cassettes reported by mKate and/or sfGFP fluorescence signals.…”

“…Plasmid pS4 was assembled with biopart assembly standard for idempotent cloning (BASIC) [ 29 , 30 ]. For complete protocol, see Storch et al.…”

Revealing the chassis-effect on a broad-host-range genetic switch and its concordance with interspecies bacterial physiologies

“…We therefore built a tractable experimental platform by transforming six Gammaproteobacteria species with a genetic toggle switch built onto a pSEVA231-derived (Kanamycin selection marker and pBBR1 origin of replication) vector (Fig. 1a, 1b) within the Biopart Assembly Standard for Idempotent Cloning (BASIC) cloning environment 29, 30 , yielding plasmid pS4. The toggle switch consists of two inducible operons acting as antagonistic expression cassettes reported by either mKate or sfGFP fluorescence signals.…”

“…pS4 toggle switch was assembled in the Biopart Assembly Standard for Idempotent Cloning 29, 30 (BASIC). For complete protocol, see Storch et al (2015).…”

Fine Tuning Genetic Circuits via Host Context and RBS Modulation

“…1a). The pVCS series was assembled using the DNA-BOT 44 platform via automated BASIC DNA assembly 45,46 , using RBSs of known translational strengths (RBS1, RBS2 and RBS3, in increasing strength) incorporated into the BASIC linkers. The core design of our toggle switches draws inspiration from the canonical Gardner et al (2000) genetic toggle switch, consisting of two antagonistic expression cassettes to create a bistable motif with each cassette being regulated by a negatively inducible promoter.…”