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. 2023 May 22;20:100677. doi: 10.1016/j.mtbio.2023.100677

Fig. 4.

Fig. 4

Pyruvoyl tetrahydropterin synthesis in E. coli ELMs. a) Schematic of aTc-inducible CRISPRa programs controlling the expression of two enzymes (GTPCH and PTPS) catalyzing the conversion of guanosine triphosphate (GTP) into the pteridine derivative pyruvoyl tetrahydropterin (PT). b) Dynamic induction cycles on E. coli ELM for PT bioproduction. Bioproduction cycles alternating between periods of induction (production ON, green), and delay (production OFF, gray), were implemented under continuous culture conditions for 15 days. Media was replenished every 24 ​h. c) PT bioproduction capacity retained following a subsequent induction cycle. PT fluorescence from ELM constructs was measured every 24 ​h, with uninduced (no aTc) and induced (+aTc) controls included in the plot. Full production capacity is defined as the maximum pteridine level achieved in Induction 1. Retained production capacity and non-baseline returns were calculated relative to the full production capacity. Values represent the mean ​± ​standard deviation from n ​= ​3–4 technical replicates. Green and gray shades denote induction and delay periods, respectively. Statistical significance in pteridine fluorescence at two consecutive time points was evaluated using a two-tailed unpaired Student's t-test (p ​> ​0.05). d) PT bioproduction in ELMs was measured following variable delays of 10 or 14 days (gray) of continuous culture before CRISPRa-programmed enzyme expressions were initiated by the addition of aTc inducer and carried out for 5 days (green). e) Persistence of bioproduction in ELMs was characterized as measured fluorescence from pyruvoyl tetrahydropterin every 24 ​h, with uninduced (no aTc) and induced (+aTc) controls included in the plot. Values represent the mean ​± ​standard deviation from n ​= ​3–4 technical replicates. Statistical significance in pteridine fluorescence at two consecutive time points was evaluated using a two-tailed unpaired Student's t-test (p ​> ​0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)