Fig. 2: The automation pipeline for high-throughput screening of CRISPRi-mediated isoprenol production provided a high success rate in building strains and highly reproducible controls.

a A suite of laboratory automation equipment facilitated the construction of sgRNA arrays. sgRNA units were dispensed with positionally dependent complementary linkers using the ECHO 550, then simultaneously digested and ligated to compose linked sgRNA units. sgRNA units were magnetically purified with an Apex KingFisher, assembled into sgRNA arrays, dispensed, and transformed into competent Escherichia coli with the MANTIS, plated using a Hamilton VANTAGE, picked with a QPix 460, and ultimately sequence validated. Validated sgRNA arrays were then dispensed with electrocompetent P. putida IY1449b (ΔphaABC, ΔmvaB, ΔhbdH, ΔldhA, ΔzwfB, ΔgntZ, and ΔliuC) cells harboring pIY670 again using the ECHO 550. Electroporations were completed in parallel using the 384-HTME prior to outgrowth and plating on a selective medium using the Hamilton VANTAGE. Assembly and transformation routinely displayed a success rate above 95% with failure most often stemming from downregulation toxicity. Strains transformed with CRISPRi arrays were adapted twice in minimal medium and cultured in a microbioreactor. Cultures were harvested for proteomics and isoprenol analysis, producing a total of ~1500 samples for the full project. This figure was partially generated using BioRender.com (https://BioRender.com/ukpmliz). b Each cycle generated 60 unique sgRNA arrays in addition to a control, which were tested in four sequential 48-well BioLector fermentations, with controls included in triplicate. c Although DBTL cycles sometimes took place months apart, isoprenol titers in control strains harboring a non-target sgRNA remained remarkably precise and reproducible, varying by 5–10% CV across all cycles. In DBTL0, the control strain was cultured across plates (n = 18) while subsequent cycles had three control strains per plate (n = 12). Longer sgRNA arrays were favored in later cycles. Error bars represent standard deviations. Source data are provided in the Source Data file.