2.1 Construction of strains and plasmids
All the strains and plasmids in this study are listed in Table S1 and
S2. E. coli DH5α was used for plasmid construction and BL21(DE3)
was chosen as the host strain. All PCR amplifications were performed by
PrimeSTAR Max DNA Polymerase (Vazyme, Nanjing, China). The plasmids
which were used to express IUP pathway were constructed on the basis of
pSU2718 (chloramphenicol-selectable). Plasmids harboring IUP under
control of constitutive promotors were constructed through restriction
enzyme digestion and ligation (KpnI , BamHI ). To construct
Plasmids harboring IUP under the control of inducible promotors, the DNA
fragments and plasmid backbone were amplified by PCR and constructed
through Gibson assembly. The constructed plasmid was introduced into
DH5α by chemical transformation or electro-transformation and correct
transformants were confirmed by colony PCR. Plasmids of IUP and enzymes
for lycopene production were introduced to BL21(DE3) through
electro-transformation.