A biofoundry workflow for the identification of genetic determinants of microbial growth inhibition

Abstract

Biofoundries integrate high-throughput software and hardware platforms with synthetic biology approaches to enable the design, execution and analyses of large-scale experiments. The unique and powerful combination of laboratory infrastructure and expertise in molecular biology and automation programming, provide flexible resources for a wide range of workflows and research areas. Here, we demonstrate the applicability of biofoundries to molecular microbiology, describing the development and application of automated workflows to identify the genetic basis of growth inhibition of the plant pathogen Streptomyces scabies by a Pseudomonas strain isolated from a potato field. Combining transposon mutagenesis with automated high-throughput antagonistic assays, the workflow accelerated the screening of 2,880 mutants to correlate growth inhibition with a biosynthetic gene cluster within two weeks.

Mammalian cell characterisation by non-invasive plate reader assay

“…Our method has the potential to greatly support advancements in the automation of mammalian cell screening both in basic and applied research. For the latter, while for several micro-organisms it is now possible to perform automated library assembly and parallel testing of high number of variants [39][40][41] , the uptake of the same workflow in mammalian cells is lagging behind. Design and assembly of large construct libraries is now possible for mammalian systems thanks to the development of toolkits that support modular and high throughput construct generation 42,43 , but the screening of such variants within their target host is slowed down by the low throughput protocols that were previously available.…”

Beyond Soil-Dwelling Actinobacteria: Fantastic Antibiotics and Where to Find Them

“…It is in this area, the less easily identified BGCs, where Pseudomonas exhibit big potential for novel chemistry discovery. In fact, the use of untargeted methods such as the generation of transposon mutant libraries of bioactive Pseu-domonas strains has led to the discovery of secondary metabolites such as the butanolide molecules styrolides A (Figure 15) and B [380], or the antimicrobial 7-hydroxytropolone (Figure 15) in two different biocontrol Pseudomonas strains [340,341], presenting this as a promising strategy to uncover useful secondary metabolites. Finally, and perhaps surprisingly, not many RiPPs have been characterized in Pseudomonas strains, with the notable exceptions of the redox cofactor pyrroloquinoline quinone (PQQ) (Figure 15) [39] and the recently discovered and highly unusual 3-thiaglutamate (Figure 15) [381].…”

“…Certain types of metabolites, such as siderophores and cyclic lipopeptides seem to be staples of this genus and are widespread in it [ 326 ], but there are also other specialized metabolites, many of them common to other often distantly related bacteria, such as Streptomyces . This shared metabolic potential can arise through horizontal gene transfer from other environmental bacteria, as seems to be the case for antibiotic byciclomycin [ 335 ] or the coronafacoyl phytotoxins [ 336 ] but also occurs through convergent evolution of separate pathways as it is the case for antibiotic fosmomycin [ 337 , 338 ] or tropolone siderophores [ 339 , 340 , 341 ].…”

“…It is in this area, the less easily identified BGCs, where Pseudomonas exhibit big potential for novel chemistry discovery. In fact, the use of untargeted methods such as the generation of transposon mutant libraries of bioactive Pseudomonas strains has led to the discovery of secondary metabolites such as the butanolide molecules styrolides A ( Figure 15 ) and B [ 380 ], or the antimicrobial 7-hydroxytropolone ( Figure 15 ) in two different biocontrol Pseudomonas strains [ 340 , 341 ], presenting this as a promising strategy to uncover useful secondary metabolites.…”

RoboMoClo: A Robotics-Assisted Modular Cloning Framework for Multiple Gene Assembly in Biofoundry

“…Recently, we established the SKy BioFoundry as a noncommercial and regional biomanufacturing platform to accelerate the strain development process (Sungkyunkwan University, South Korea). Compared to the previous biofoundries , (MIT-Broad Foundry, MA; iBioFAB, Illinois; DOE Agile BioFoundry, CA; London DNA Foundry, UK; Edinburgh Genome Foundry, UK; Earlham Biofoundry, UK; Forschungszentrum Jülich GmbH, Germany; TIB, CAS, China; etc. ), the SKy BioFoundry lacks a centralized robotic arm to perform fully automated processes connecting process units but has a semiautomated workflow to connect each process unit on the deck of the two liquid handler platforms with a robotic manipulator arm (Evo200, Tecan).…”