The primer pairs used in the cloning strategy are detailed in Table . The vector backbone (pET28-ptetO::gfpv2) was linearized by PCR amplification. The micABCDE fragment was PCR-amplified with homology tails for pET28-ptetO::gfpv2 but a restriction site for PmlI (NEB) was added to the 3' end of the amplicon to allow linearization of the large pET28-ptetO::mi-cABCDE-gfpv2 construct through endonuclease digestion. The micFG fragment was obtained through PCR with homology tails to ptetO::micA-BCDE-gfpv2, which enabled assembly through SLIC. The ptetO::micA-BCDEFG-gfpv2 construct was then used for heterologous expression (Fig. ). Amplification of all fragments was carried out as standard 50 µL PCR reactions with a final concentration of 1× Q5 High-Fidelity 2× Master Mix (NEB), 0.5 µM of forward and reverse primer and 50-100 ng of DNA template using a Veriti TM Thermal Cycler. Thermal cycling programs varied according to template and were as follows: initial denaturation 98 ºC for 30 sec; denaturation 98 ºC 10 sec; annealing at 62 ºC for linearization of vector backbone pET28-ptetO::gfpv2 or 63 ºC for amplification of the micABCD amplicon or 57 ºC for amplification of the micEF amplicon; 4) extension at 72 ºC, 6min for pET28-ptetO::gfpv2, or 8 min for both micABCD and micFG, followed by a final extension of 10 min at 72 ºC. The PCR product of vector pET28-ptetO::gfpv2 was first treated with DpnI (NEB) (37 ºC 1 h and 65 ºC for 20 min) in order to remove template plasmid that could act as transformation background. Prior to cloning, all linear fragments were purified by gel excision using the NZYGelpure kit (NZYTech) and eluted in 30 µL. For the construction of the ptetO::micABCDE-gfpv2, the SLIC method was performed using 1× Buffer 2.1 (NEB), 0.5 µL of T4 polymerase (NEB), a variable volume of linear DNA fragments for assembly (concentrations calculated as described by Greunke et al , ranging from 0.02-0.5 pmol) and molecular biology-grade water, in a total reaction volume of 10 µL. The SLIC reaction mixture was incubated for 30 s at 50 ºC and then for 10 min on ice. A total of 5 µL of reaction mixture was transformed by heat shock into chemically competent E. coli TOP10 cells. The presence of positive clones was evaluated by colony PCR (Table ), using a primer pair amplifying a portion of the ptetO::micA region. Clones leading to amplicons of the expected size were grown overnight at 37 ºC, in liquid LB medium supplemented with 50 µg mL -1 of kanamycin, with shaking at 180 rpm. Plasmids were isolated from overnight cultures using the NZYMiniprep kit (NZYTech) and sent for sequencing to confirm the integration site.