All plasmid constructions were conducted in E. coli DH5α through transformation using the heat shock method. The chemically competent E. coli cells were prepared using the Inoue method (15). To dynamically control biofilm formation and dispersal, a bidirectionally controllable c-di-GMP module, comprising a previously constructed NIR light–responsive c-di-GMP gene module (i.e., bphS and bphO) (14, 16) and a blue light–activated PDE gene eb1 (17), was constructed as follows. The gene eb1 with a Ptac promoter and the Shine-Dalgarno (SD) sequence was amplified by polymerase chain reaction (PCR) from pMal-EB1 using primers Blue-Ptac-F (5′-GGACTAGTTGACAATCATCGGCTCGTAT-3′) and Blue-Ptac-R (5′-GGACTAGTTCATGAGTCCAGACTGATGGTTC-3′) (Spe I enzyme site underlined). The Spe I–digested fragment was ligated with the pYYDT-L fragment, which was cut using Spe I for fragment ligation and dephosphorylated using FastAP thermosensitive alkaline phosphatase, forming the plasmid pYYDT-RB. The constructs were selected by colony PCR using YYD-F (5′-GCCTCAGGCATTTGAGAAGCACA-3′) and YYD-R (5′-AGAGCGTTCACCGACAAACAACAGATAA-3′) and further verified to confirm the orientation of eb1 by DNA sequencing (AITbiotech, Singapore). In pYYDT-RB, bphS and bphO were placed under Ptac as an operon, whereas eb1 with an additional Ptac promoter was inserted downstream of the bphS-bphO operon to achieve coordinated efficient expression of all genes. The gene aiiO encoding a QQ enzyme (10) was then combined with bidirectionally controlled c-di-GMP module in E. coli BL21(DE). A 99–base pair (bp) fragment, flanked by Nru I restriction sites at each end, was amplified by PCR from the multiple cloning sites of pUCP22 (18, 19). The Nru I–digested 99-bp fragment was inserted at an Nru I site, near the C-terminal, of aiiO to generate an aiiO mutant (i.e., maiiO) that does not produce QQ enzyme. The aiiO gene (10) and its mutant maiiO were cloned into pTrcHis2-aiiO and pTrcHis2-maiiO, respectively, with ampicillin- and kanamycin-resistant markers (10). The mCherry gene with a PguaB promoter and the SD sequence, flanked by Pme I restriction sites at each end, was amplified by PCR from pABG5:PguaB-mCherry. The Pme I–digested PguaB.mCherry fragment was inserted at a Pme I site downstream of the kanamycin resistance gene in both pTrcHis2-aiiO and pTrcHis2-maiiO to construct the pTrcHis2-aiiO.mCherry and pTrcHis2-maiiO.mCherry, respectively. To enable the compatibility of pTrcHis2-aiiO.mCherry and pTrcHis2-maiiO.mCherry with pYYDT-RB in a stable double plasmid system, the kanamycin resistance genes flanked by two Hind III restriction sites at each end were removed using Hind III restriction enzyme, and then pTrcHis2-aiiO.mCherry and pTrcHis2-maiiO.mCherry were recircularized by T4 ligase to generate the resulting plasmids pAiiO and pmAiiO. pYYDT-RB and pAiiO (or pmAiiO) were simultaneously transformed into E. coli BL21(DE). The sequence details for the constructed gene circuits are included in table S1.

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