Mops ez rich defined medium

The culture medium lysogeny broth (LB) was used for cloning purposes. Transformed cultures were plated out on lysogenic broth agar (LBA) and grown overnight at 30 °C. If required, LB and LBA medium were supplemented with appropriate antibiotics (100 μg/mL ampicillin or 50 μg/ml kanamycin). For microscopy and the indirect analysis method, strains were grown in minimal medium glucose (MMGlc). If using DH10B cells, 0.4% casamino acids were added. If required, the culture medium was supplemented with appropriate antibiotics and inducers (0–1% L-arabinose or 0–0.2 mM IPTG). For fluorescence experiments with the smart library of sRNAs, strains were grown in MOPS EZ Rich Defined medium (Teknova) with 100 μg/mL ampicillin and 2% glucose as carbon source. For all fluorescent measurements, independent of medium (EZ Rich or MMGlc) used, precultures were grown overnight on a Compact Digital Microplate Shaker (Thermo Fisher Scientific) at 30 °C and 800 rpm, in 96-well flat bottomed microtiter plates (Greiner Bio-One), in 150 μL LB per well with appropriate antibiotics, enclosed by a Breathe-Easy sticker. Precultures were used for 1/150 dilution in 150 μL of defined medium (EZ Rich or MMGlc) containing a specific concentration of the inducer, and were grown for 24 h at 30 °C and 800 rpm unless stated otherwise.

Escherichia coli DH10b (F– mcrA
Δ(mrr-hsdRMS-mcrBC)
Φ80lacZΔM15 ΔlacX74 recA1
endA1 araD139 Δ(ara leu)
7697galU galKrpsLnupG
λ–) (Durfee et al, 2008 (link)) was
used for cloning (New England Biolabs, MA, C3019). Escherichia coli K-12
MG1655* [F-λ-ilvG- rfb-50 rph-1 Δ(araCBAD) Δ(LacI)]
(Blattner et al, 1997 (link)) was used for
measurement experiments. Cells were grown in LB Miller broth (Difco, MI, 90003-350) for overnight
growth and cloning, and MOPS EZ Rich Defined Medium (Teknova, CA, M2105) with 0.4% glycerol
carbon source for measurement experiments. Ampicillin (100 μg/ml), kanamycin (50
μg/ml), and spectinomycin sulfate (50 μg/ml) were used to maintain plasmids. Arabinose
(Sigma Aldrich, MO, A3256), 2,4-diacetylphloroglucinol (Santa Cruz Biotechnology, TX, CAS
2161-86-6), and anhydrotetracycline (aTc) (Sigma Aldrich, MO, 37919) were used as chemical inducers.
The fluorescent protein reporters YFP (Cormack et al, 1996 (link)) and mRFP1 (Campbell et al, 2002 (link)) were measured with cytometry to determine gene expression.

Escherichia coli NEB 10‐beta (C3019I, New England BioLabs, Ipswich, MA, USA) was used for all routine cloning. All genetic circuit measurements were done using E. coli K‐12 MG1655 * [F‐ λ‐ ilvG‐ rfb‐50 rph‐1 Δ(araCBAD) Δ(LacI)] (Blattner et al, 1997 (link); Nielsen & Voigt, 2014 (link)). Cells were grown in in MOPS EZ Rich Defined Medium (Teknova, M2105) with 0.2% glucose (Teknova, G0520). Kanamycin (50 µg/ml, GoldBio, K‐120‐5) was used to maintain plasmids. Chemical inducers used the following: vanillic acid (Van) (Millipore Sigma, 94770); isopropyl β‐d‐1‐thiogalactopyranoside (IPTG) (GoldBio, I2481C); anhydrotetracycline (aTc) (Millipore Sigma, 37919); and choline chloride (Chol) (Millipore Sigma, C7017). DNA oligos and genes were ordered from Integrated DNA Technologies (Coralville, IA) and Twist Biosciences (San Francisco, CA). All plasmids were constructed from the parental pDAA038 backbone (Appendix Table S3) using TypeIIS assembly to insert circuit components between BsaI sites. A table of genetic parts and full plasmid sequences are provided in Appendix Table S2 and S3. Key plasmid maps are shown in Appendix Fig S5.

Overnight cultures of E. coli strains were diluted by 1:100 in MOPS EZ-rich defined medium (Teknova). 0.2% glucose was used as the carbon source for imaging Hfq-mMaple3 WT and mutants under NT and rifampicin treated conditions. 0.2% fructose was used as the carbon source with 100 µg/mL ampicillin for cases with sRNA overexpression, mMaple3 control, mMaple3 fused sAB-70, and mMaple3 fused scFv-GCN4. Cultures were grown at 37°C aerobically. Plasmid-encoded sRNAs were induced by 1 mM IPTG when the OD₆₀₀ of the cell culture was ~0.1. Induced cells were grown for ~45 min before imaging. Plasmid-encoded mMaple3 protein (with 100–400 μM IPTG), mMaple3 fused sAB-70 (with 1 mM IPTG), and mMaple3 fused scFv-GCN4 (with 1 mM IPTG) were expressed and imaged in the same way. For the rifampicin treatment, rifampicin was added to a final concentration of 200 μg/mL when the OD₆₀₀ of the cell culture was ~0.2, and the cells were incubated for 15 min before imaging.

All strains and plasmids used in this study are derivatives of E. coli K-12 strain MG1655 (rph-1) and are listed in Supplemental Table S1. Primers used for strain construction are listed in Supplemental Table S2. Strain construction is described in Supplemental Materials and Methods.
All strains were grown in liquid medium or agar plates containing either Lennox broth (LB), M9 minimal medium supplemented with 0.001% vitamin B1 and 0.2% glucose or succinate, or MOPS EZ rich defined medium (Teknova) supplemented with 0.4% glycerol instead of glucose. Antibiotics were used in the following final concentrations: ampicillin, 100 µg/mL; kanamycin, 25 µg/mL; chloramphenicol, 25 µg/mL or 10 µg/mL (for any mutant(s) containing hfq deletion), tetracycline, 12.5 µg/mL, zeocin, 25 µg/mL, and rifampicin, 250 µg/mL. 2, 2′ dipyridyl was added to liquid medium at a final concentration of 250 µM. All liquid cultures and bacteria on solid medium were grown aerobically at 37°C. Overnight cultures were diluted 1:200 fold in appropriate medium and grown until desired densities were reached. Growth was determined by measuring the optical densities of liquid cultures at 600 nm (OD₆₀₀). Cultures were considered to be in exponential phase when they reached OD₆₀₀ between 0.3 and 0.4 in LB or OD₆₀₀ of ∼1.0 in MOPS EZ rich defined medium.