pBAD–LCY

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	Double transformants were grown in LB medium under induction with 0.2% arabinose and selection with chloramphenicol and ampicillin at 28°C and 250 rpm to an OD600 between 0.5 and 1.0.	Then, 10 ml culture per sample were harvested by centrifugation (5000 g, 4 min, 4°C), the supernatant completely removed and the cell pellet stored at −20°C until further analysis.

Protocol tips
Double transformants were grown in LB medium under induction with 0.2% arabinose and selection with chloramphenicol and ampicillin at 28°C and 250 rpm to an OD600 between 0.5 and 1.0.
Downstream tips
Then, 10 ml culture per sample were harvested by centrifugation (5000 g, 4 min, 4°C), the supernatant completely removed and the cell pellet stored at −20°C until further analysis.

Publication protocol

For construction of plasmid pLYCO2, the crtZ gene in plasmid pACCARΔ25crtX (Kajiwara et al., 1995) was excised by double digest with AatII and NsiI, the overhangs filled using Phusion® High‐Fidelity DNA Polymerase (NEB; https://www.neb.com/) and religated yielding plasmid pBETA2. Double digest of pBETA2 with AsiSI and SnaBI removed crtX and crtY, and filling of overhangs and religation yielded plasmid pLYCO2. For functional analyses, E. coli TOP10 were co‐transformed with pLYCO2 and either of the various pBAD–LCY constructs. Double transformants were grown in LB medium under induction with 0.2% arabinose and selection with chloramphenicol and ampicillin at 28°C and 250 rpm to an OD600 between 0.5 and 1.0. Then, 10 ml culture per sample were harvested by centrifugation (5000 g, 4 min, 4°C), the supernatant completely removed and the cell pellet stored at −20°C until further analysis.

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