Porcine hemin

We used BHIS as the base medium for this study, to match growth conditions during the initial construction of the transposon pool [26 (link)]. Our BHIS formulation is hydrated Brain Heart Infusion powder (Difco, Cat. #237200) supplemented with 0.2% (w/v) sodium bicarbonate (Fisher bioreagents, Cat. #BP328-500), 0.05% (w/v) porcine hemin (Alfa Aesar, Cat. #A11165), and, in some cases, 0.1% (w/v) L-cysteine (Thermo Scientific, Cat. #A10435.18). In a previous study, we found that exogenous cysteine led to the production of H₂S by B. theta VPI-5482 [30 (link)]. With the large volumes required for this approach, the H₂S levels were enough to saturate our hydrogen sulfide scrubbing column and make working with the collection dangerous. Therefore, for all steps that required growth of the collection in substantial volumes, we did not include cysteine in the BHIS formulation. Cultures were incubated in a custom anaerobic chamber (Coy Laboratories) using an 85% nitrogen–10% carbon dioxide–5% hydrogen anaerobic gas mix (Praxair, Cat. #BI NICDHYC4-K).

Sf21 lysate preparation and coupled CFPS have already been described and explained (Brödel et al., 2013b (link); Quast et al., 2015 (link)). Minor changes were made during lysate preparation. In all buffers used during the lysate preparation, DTT was omitted, and 0.05 mM GSH and 0.25 mM GSSG were added. Coupled transcription-translation reactions were performed in a batch mode format. Protein synthesis was performed in a thermomixer (Thermomixer comfort, Eppendorf, Hamburg, Germany) at 21°C (if not stated otherwise) and with gentle shaking at 500 rpm for 3 h. Reaction volumes of 50 μL were composed of 40% (v/v) Sf21 cell lysate, 100 µM of each canonical amino acid, nucleoside triphosphates (1.75 mM ATP, 0.30 mM CTP, 0.30 mM GTP, and 0.30 mM UTP), 100 ng/μL vector DNA, and 1 U/µL T7 RNA-polymerase (Agilent, Waldbronn, Germany), 0–75 µM porcine hemin (Alfa Aesar, Haverhill, USA) dissolved in NaOH. To monitor protein quality and quantity, reaction mixtures were supplemented with ¹⁴C-labeled leucine (Perkin Elmer, Inc.; Baesweiler, Germany). No template controls (NTC) were prepared in the same way as the samples except that the DNA template was replaced by water.