Hifi gibson assembly master mix

The inducible pJV-Tyr1 plasmid containing Priestia megaterium (formerly Bacillus megaterium) tyrosinase (Tyr1) used in this study was previously published by our group (Wang et al., 2020 (link)). The sequence of Rhizobium etli melA gene was obtained from the NCBI database and synthesized by Eurofins. The E. coli hpaBC gene sequence in the plasmid pRSF1010 was kindly provided by Dr. Nathan Schwalm at Army Research Laboratory. The hpaBC genes are expressed in tandem from a single operon. Using Q5 High Fidelity Polymerase (New England Biolabs), the melA and hpaBC gene sequences were amplified with primer pairs MelA-pJ_F/MelA-pJ_R and HpaBC-pJ_F/HpaBC-pJ_R, respectively. The pJV298 vector backbone was amplified with primer pairs pJV_melA_F/pJV_melA_R and pJV_hpaBC_F/pJV_hpaBC_R for MelA and HpaBC, respectively. The genes for melA and hpaBC were cloned into the pJV298 plasmid backbone in a similar manner as was done previously for tyr1 using the HiFi Gibson Assembly Master Mix (New England Biolabs) (Wang et al., 2020 (link)).

The pCB1D5-GFP plasmid (DVC) (Tschirhart et al., 2019 (link)) was used as the backbone for initial cloning of the tyr1, hpaBC, and melA genes. The pCB1D5 plasmids contain the constitutive Anderson promoter J23106 and RBS B0032 upstream of the cloned genes, and was constructed using the HiFi Gibson Assembly Master Mix (New England Biolabs) (iGEM, 2023 ). The vector was amplified using Q5 High Fidelity Polymerase (New England Biolabs) with the primer pairs pCB1D5_tyr_F/pCB1D5_tyr_R, pCB1D5_hpaBC_F/pCB1D5_hpaBC_R, and pCB1D5_melA_F/pCB1D5_melA_R for Tyr1, HpaBC, and MelA assembly, respectively. The tyr1, hpaBC, and melA genes were amplified using primer pairs Tyr1-pC_F/Tyr1-pC_R, HpaBC-pC_F/HpaBC-pC_R, and MelA-pC_F/MelA-pC_R, respectively. The Gibson DNA assembly reaction was set up and run as indicated by the manufacturer’s protocol, and 2 µL was transformed into NEB10β chemically competent cells as per manufacturer’s instructions.

A DNA fragment consisting of BBa_J23119 promoter-Bujard RBS-mRFP1-BBa_B00015 terminator (termed mRFP1 reporter, Table 2) was designed using parts from the Repository of Standard Biological Parts (http://parts.igem.org) and synthesized by Integrated DNA Technologies. Plasmid pBBRMCS1-5 was amplified with primers oCAH16 and oCAH17 and assembled with the synthetic mRFP1 reporter fragment using HiFi Gibson Assembly Master Mix (New England Biolabs) to generate the plasmid pDS1. DNA fragments containing an Anderson series promoter-Bujard RBS-mRFP1 were amplified from BBa_J61002 supplied with the iGEM 2021 distribution kit using primers oCAH1194 and oCAH1195 and assembled with pDS1 amplified with primers oCAH1196 and oCAH16 to generate a BHR Anderson promoter-probe plasmid series. Notably promoter parts BBa_J23103, BBa_J23108, BBa_J23109, BBa_J23111, BBa_J23112 were not constructed here either because they exhibit limited activity in E. coli or they exhibit redundant activity with other promoters in the series. E. coli DH10B, M. capsulatus Bath, or M. trichosporium OB3b harboring the Anderson promoter-probe series were cultivated in liquid medium containing 10 μg/mL gentamicin to ∼ OD₆₀₀ 1.0; 200 μL culture was transferred to a 96-well microplate, and mRFP1 fluorescence (ex_532nm, em_588nm, gain = 80) and optical density (A_600nm) was measured with a BioTek Synergy Mx microplate reader.