Mops ez rich

The Escherichia coli DH10B derivative NEB 10‐beta Δ(ara‐leu) 7697 araD139 fhuA ΔlacX74 galK16 galE15 e14‐ϕ80dlacZΔM15 recA1 relA1 endA1 nupG rpsL (StrR) rph spoT1 Δ(mrr‐hsdRMS‐mcrBC) was used for cloning and measurements (New England Biolabs, MA, C3019). Cells were grown in LB Miller broth (Difco, MI, 90003‐350) for harvesting plasmids. Cells were grown in MOPS EZ Rich (Teknova, M2105) defined medium with 0.2% glycerol for circuit performance measurements. To select for the presence of plasmids, 50 μg/ml kanamycin (Gold Biotechnology, MO, K‐120‐5) and 50 μg/ml spectinomycin (Gold Biotechnology, MO, S‐140‐5) antibiotics were used.

Cell cultures were grown from an individual colony in LB Miller medium (LB) to the exponential phase with an appropriate selection of antibiotics. Strains with plasmids were grown with 50 μg/ml carbenicillin during overnight growth. For microscopy, cells were then washed and diluted into LB, MOPS Rich Defined Medium (RDM; MOPS EZ Rich Defined Medium, M2105, Teknova), or defined MOPS minimal medium (MM) (MOPS Minimal Media Kit, M2106, Teknova) without antibiotic. We added 0.02–0.4% (w/v) glucose to RDM and MM, depending on the experiment (Appendix Tables S1 and S2). Cells were grown at 30°C in liquid media in a shaking incubator. To monitor single‐cell growth without division, we inhibited cell division either by inducing sulA with 1 mM IPTG from plasmid pDB192 or by adding 10 μg/ml aztreonam (Sigma‐Aldrich, A6848) shortly before microscopy (Appendix Tables S1 and S2). To inhibit cell wall synthesis, we treated cells with vancomycin (Fisher Scientific, BP2958‐1), Fosfomycin (Sigma‐Aldrich, P5396‐1G), or D‐cycloserine (Sigma‐Aldrich, 30020‐1G). To perform microscopy, we used three different supports, flow chambers, agarose pads, or donut‐shaped chambers, as described in the following. During sample preparation and subsequent imaging, the temperature was maintained at 30°C.