Extended Data Fig. 4. Effect of induction on viability and OD₆₀₀ for bARG_Ser-expressing EcN in liquid culture.

(a-b) Colony forming units (CFU) per mL of culture (a) and optical density at 600 nm (b) during daily sub-culturing into LB media with 25 μg/mL chloramphenicol (+chlor), without chloramphenicol (-chlor), or without chloramphenicol and with 0.1% (w/v) L-arabinose (-chlor +L-ara) using pBAD-bARG_Ser-AxeTxe EcN. Curves represent the mean of n = 4 biological replicates. (c-d) Colony forming units (CFUs) per mL of culture on chloramphenicol plates (c) and optical density at 600 nm (p values from left to right: 0.0638556, 1.019913e-6, 0.0217493, 3.800448e-6) (d) of pBAD-bARG_Ser-AxeTxe EcN and pBAD-FP-AxeTxe EcN cultures 24 hours after sub-culturing into LB media with the same conditions as in (a-b) and in Fig. 2c. Asterisks indicate statistical significance by two-tailed, unpaired Student’s t-tests (**** = p < 0.0001, * = p < 0.05, ns = no significance); n = 4 biological replicates. (e) OD₆₀₀ versus time after inducing pBAD-bARG_Ser-AxeTxe and pBAD-FP-AxeTxe EcN strains with 0.1% (w/v) L-arabinose in liquid culture at 37 °C (n = 4 biological replicates). Between 5 and 24 hours post-induction, when the OD₆₀₀ of all cultures decreased, the OD₆₀₀ of FP-expressing cultures was slightly higher than that of the bARG_Ser-expressing cultures, likely due to expression of red fluorescent protein which is known to absorb light at 600 nm⁸⁶. (f) Representative image of colonies from the experiment in Fig. 2c on chloramphenicol plates with (right) and without (left) 0.1% L-arabinose. The opacity of the colonies on plates with L-arabinose indicates bARG_Ser expression and was used to screen for mutants deficient in bARG_Ser expression (see Supplementary Fig. 7). (g-h) Representative phase contrast microscopy (from n = 4 biological replicates) (g) and transmission electron microscopy (from n = 23 images taken of a single sample) (h) images of pBAD-bARG_Ser-AxeTxe EcN cells grown on plates with 0.1% (w/v) L-arabinose at 37 °C. Scale bars are 10 µm (g) and 500 nm (h). Curves and lines represent the mean for a-e.

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