pCcrtB

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Upstream tips	Protocol tips	Downstream tips
To assess whether KgCrtB can be expressed in E. coli, we cloned the KgCrtB gene into the NdeI/HindIII site of the pColdI vector (TaKaRa, Japan).	To synthesize lycopene in E. coli, the two plasmids constructed (pCcrtB and pRSCrtEI) were co-transformed and co-expressed in BL21 (DE3) at 37°C for 24 h culture.

Upstream tips
To assess whether KgCrtB can be expressed in E. coli, we cloned the KgCrtB gene into the NdeI/HindIII site of the pColdI vector (TaKaRa, Japan).
Protocol tips
To synthesize lycopene in E. coli, the two plasmids constructed (pCcrtB and pRSCrtEI) were co-transformed and co-expressed in BL21 (DE3) at 37°C for 24 h culture.

Publication protocol

"To assess whether KgCrtB can be expressed in E. coli, we cloned the KgCrtB gene into the NdeI/HindIII site of the pColdI vector (TaKaRa, Japan). We amplified the genes by using primers (Table 1) that had an NdeI at the N-terminus and a HindIII site at the C-terminus, digested the product by using restriction enzymes,
subcloned it into the pColdI vector, and called this plasmid pCcrtB. In addition, to synthesize lycopene in E. coli, we cloned the P. haeundaensis crtE and crtI genes in the MCS1 and MCS2 sites, respectively, of the pRSFDuet-1(Novagen, USA) coexpression vector. To clone the crtE (GGPP synthase) gene into MCS1, we designed an EcoRI restriction site and a NotI restriction site at the N- and C- terminal, respectively, and to clone the crtI (phytoene desaturase) into MCS2, we designed an NdeI restriction
site and XhoI restriction site at the N- and C- terminal, respectively. The completed recombinant plasmid was named pRSCrtEI. Following this, the selection of transformed E. coli was conducted
by ampicillin resistance. Transformed E. coli was cultured in LB medium containing 50 µg/ml of ampicillin at 37°C until OD600 reached 0.5. Following this, 0.1 mM IPTG was added at 16°C to induce the overexpression of CrtB. To analyze the expression level, 12% SDS-PAGE was conducted. The proteins were electrotransferred from the SDS-PAGE gel to a nitrocellulose membrane, probed with goat antiserum against 6-His tag, and incubated with alkaline phosphatase coupled to goat antibody
against goat IgG. The nitrocellulose membrane was developed using BCIP/NBT (Sigma-Aldrich, USA) [27]. Separately, the crtE and crtI genes from P. haeundaensis were inserted into the pRSFDuet-1 vector (Novagen, USA) to synthesize lycopene in E. coli BL21 (DE3). The resulting pRScrtEI plasmid was transformed into E. coli BL21 (DE3) followed by a kanamycin selection process.
To synthesize lycopene in E. coli, the two plasmids constructed (pCcrtB and pRSCrtEI) were co-transformed and co-expressed in BL21 (DE3) at 37°C for 24 h culture."

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