Golden Gate cloning of Level 0 standard parts

All genetic parts were cloned as individual Level 0 standard modules into the universal Level 0 acceptor plasmid pAGM9121 (Spectinomycin^R). Therefore, three functional units were pre-defined: (a) signal peptide (contains start codon); (b) gene (lacking start and stop codon) and (c) C-terminal Protein-tag (contains stop codon). 4 bp sticky overhangs that are released upon Type II s enzyme treatment (BsaI and BbsI) and guide subsequently a correct reassembly were chosen accordingly to the nomenclature of gene assembly as described within the ModularCloning (MoClo) system³³. An overview of the reassembly concept is provided in Supplementary Fig. 1. For the cloning of the individual modules suitable oligonucleotides were designed to allow for cloning into pAGM9121. Primers followed a general scheme (Supplementary Fig. 1). Fragments were amplified by PCR from a suitable template sequence or generated by hybridisation of two complementary oligonucleotides. PCR products were analysed as small aliquot (5 µL) by agarose gel electrophoresis for occurrence of the expected size and the remaining sample subsequently recovered and purified using a NucleoSpin^® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, DE). Golden Gate reactions were performed in a total volume of 15 µL. The final reaction volume contained 1-fold concentrated T4 ligase buffer (Promega, Madison, US). Prepared reaction mixtures containing ligase buffer, acceptor plasmid (20 fmol) and the corresponding insert (20 fmol) was adjusted to 13.5 µL with ddH₂O. In a final step, the corresponding enzymes were quickly added. First, a volume of 0.5 μL of the respective restriction enzyme BbsI (5 units/µL) was added and then 1 μL (1–3 units/µL) of T4 ligase. Golden Gate reactions were performed for 3 h (37 °C) and concluded by an additional enzyme inactivation step (80 °C; 20 min). The whole Golden Gate reaction volume was used to transform chemically competent E. coli DH10B cells. After heat shock transformation and recovery, the mixture was plated in different quantities on selective LB Agar plates (50 μg × mL^-1 X-Gal; 100 μg × mL⁻¹ Spectinomycin; 150 μM IPTG). Based on the occurrence of the lacZ selection marker one can easily distinguish between white colonies (recombined plasmid) and empty plasmid (blue). In general, the described protocol led to several thousand recombinant colonies with a nearly absolute proportion (>99%) of recombined, white colonies. Single colonies were checked for correct insert sizes by means of colony PCR (pAGM9121 sequencing primer; Supplementary Table 1). Positively identified clones were inoculated into 4 mL of TB-Medium (100 μg × mL⁻¹ Spectinomycin) and corresponding plasmid DNA prepared (NucleoSpin Plasmid Kit (Macherey-Nagel, Düren, DE)). After verification of the correct, intended insert sequence by Sanger Sequencing (Eurofins Genomics, Ebersbach, DE) respective plasmids were included for further use within the modular Golden Gate cloning approaches.