Figure 1: The workflow of the synthetic biology strategy used to activate a cryptic biosynthetic gene cluster. | Nature Communications

Figure 1: The workflow of the synthetic biology strategy used to activate a cryptic biosynthetic gene cluster.

From: Activation and characterization of a cryptic polycyclic tetramate macrolactam biosynthetic gene cluster

Figure 1

Key steps include: (a) analysing a sequenced genome or metagenome to identify target biosynthetic gene clusters; (b) PCR-amplifying or chemically synthesizing the corresponding DNA fragments; (c) selecting a set of suitable promoters (well-characterized or newly identified according to the target heterologous host) for gene cluster reconstruction; (d) assembling the reconstructed biosynthetic gene cluster in yeast; (e) isolating the assembled constructs from yeast and re-transforming them into E. coli for plasmid enrichment and verification; (f) verifying the assembled constructs by restriction digestion; (g) transferring the reconstructed biosynthetic gene cluster into the target heterologous host; (h) growing the strains in either liquid or solid media; (i) analysing samples on HPLC for product detection; (j) characterizing the potential products by LC-MS and NMR.

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