Fig. 1: Automated construction of 88 distinct plasmids coding for lycopene pathway operons containing genes in different orders, with varying promoters and RBSes.

a Plasmids coding for lycopene genes were assembled using the BASIC method (with DNA linkers). Genes in the lycopene pathway, crtE, crtB, and crtI (parts 3, 4, and 5), were assembled in an operon with UTR-RBS linkers containing different RBSes. The promoter (part 2) and the 3-gene operon were assembled into a backbone with p15A origin of replication (ORI) and chloramphenicol resistance gene (Cmp-R). The assembled parts were flanked by methylated linkers that recapitulate BASIC prefix and suffix (LMP and LMS). b Number of constructs with successful transformant colonies and their color are reported from Micalis Institute, Paris, and Imperial College, London. Number of constructs common to both laboratories are in the intersection. Data are from spotting 10 µL of transformation reactions by Opentrons (left) and from spotting 100 µL manually or 40 µL by Opentrons (right) on LB plates. c Count-plots show the number of constructs with successful colonies, grouped by position and gene (details in Supplementary file ‘Dataset 1’). Results are from Paris (left), from London (right), and in common (middle). Constructs have a weaker promoter (top) or stronger promoter (bottom). The RBSes are differentiated by colors. The genes’ positions in the operon are indicated on the x-axis. Means of the number of constructs for each promoter are shown by dashed lines. d Lycopene measurement (mg of lycopene per gDCW) from different constructs from both laboratories. Types of RBSes and promoters, and gene orders are indicated. E: crtE, B: crtB, I: crtI. Promoters and terminators are shown at the extremities. e Examples of red and white colonies (top), pellet preparation (middle) and acetone extracted lycopene (bottom). Source data are provided in the ‘Source Data’ file.