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. 2020 Jul 25;13(6):1793–1806. doi: 10.1111/1751-7915.13609

Fig. 8.

Fig. 8

Overview of the vioABDE operon construction via SevaBrick Assembly.

A. Step 1: Convergence of each CDS to the repository vector pSB1C3. Each CDS was amplified with unique convergence primers, following the strategy described in the part convergence paragraph, and later cloned into pSB1C3. Step 2: PCR amplification of each particular CDS using the OP primers (OP1‐OP4) to introduce the RBS BBa_B0034 and unique sticky ends for directional cloning of all CDS in the order of vioA‐vioB‐vioD‐vioE. Step 3: All RBS‐CDS amplicons were assembled with either (i) a linear pSEVAb23BBa_J23105 vector resulting in the constitutive vioABDE operon or (ii) a pre‐linearized with Ev/Pv pSEVAb23 vector and the pre‐amplified with Ep/Sp XylS/Pm expression system, resulting in the inducible vioABDE operon.

B. Cells transformed with the constitutive vioABDE operon (left) and the inducible XylS/Pm‐vioABDE operon (right) form black colonies due to the production of proviolacein.