Strains
T7 Express Escherichia coli (New England Biolabs, C2566) were used for MFE and ODMR experiments in Figs. 1–3. E. coli MG1655 cells were used for multiplexing and mother machine lock-in experiments in Fig. 4, owing to its effectiveness as a heterologous gene expression strain, and past optimization of mother machine chip loading protocols for this host. MG1655 was also used for Fig. 5. NEB Dh5α E. coli was used for plasmid production and genetic engineering steps, but not experimental data collection.
Cell media
TB auto-induction medium (Formedium, AIMTB0260) was used for growth of liquid cultures of T7 Express strains. LB medium (Formedium, LBX0102) was used for growth of MG1655 strains in liquid cultures and agar plates (Formedium, AGR10). M9 medium (Formedium, MMS0102; supplemented with 0.2 g l−1 Pluronic F-127 (Sigma Aldrich, P2443-250G), 0.34 g l−1 thiamine hydrochloride, 0.4% glucose, 0.2% casamino acids, 2 mM MgSO 4 , 0.1 mM CaCl 2 , 50 mg l−1 EDTA Na 2.2 H 2 0, 5 mg l−1 FeCl 3 , 0.84 mg l−1 ZnCl 2 , 0.13 mg l−1 CuCl 2.2 H 2 O, 0.05 mg l−1 CoCl 2 , 0.1 mg l−1 H 3 BO 3 and 0.016 mg l−1 MnCl 2.4 H 2 O) was used for mother machine experiments. Antibiotic stocks were prepared as follows: 100 mg ml−1 carbenicillin (Formedium, CAR0025) dissolved in 50% (v/v) ethanol and 20 mg ml−1 chloramphenicol (Fisher, 10368030) dissolved in 100% (v/v) ethanol; stocks were diluted 1,000× for experiments.
Plasmid construction
Whole plasmid sequencing was performed by Plasmidsaurus using Oxford Nanopore Technology.
Derivative plasmids of those generated by directed evolution (for example, MagLOV + mCherry) were constructed using the EcoFlex kit (Addgene kit 1000000080) following standard EcoFlex protocols61. Level 1 assemblies were performed using NEB BsaI-HFv2, NEB T4 DNA Ligase Reaction Buffer (B0202S) and Thermo Scientific T4 DNA Ligase (EL0011). Level 2 assemblies were performed using NEBridge Golden Gate Assembly Kit BsmBI-v2 and NEB T4 DNA Ligase Reaction Buffer (B0202S).
Level 1 PCR cycling protocol: 30× (5 min at 37 °C → 5 min at 16 °C) → 10 min at 60 °C → 20 min at 80 °C.
Level 2 overnight PCR cycling protocol: 65× (5 min at 42 °C → 5 min at 16 °C) → 10 min at 60 °C → 20 min at 80 °C.
pTU1 was used as a negative control plasmid (Supplementary Note 2); it is part of the EcoFlex kit.
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