3.1 Construction of the bicistronic T7 expression system
The artificial bicistronic structure in prokaryotes has been shown to
enhance the production of recombinant
proteins.[20] In this study, to further improve
the expression performance of the T7 promoter in E. coli , we
constructed the T7 in a bicistronic manner. We selected 8 well-performed
fore-cistrons from the Pbtac system based on our
previous study in C. glutamicum ,[19] and
optimized their sequences according to the codon preference of E.
coli , thus generating 8 additional sequences. In total, 16 fore-cistron
sequences were used to construct bicistronic T7 systems, and the
sequence information was presented in Table 1 . According to the
characteristics of the BCD expression cassette, a conserved SD sequence
(the second SD motif, SD2) and a translation coupling frame were
inserted between the fore-cistron and the reporter gene EGFP
(Fig. 1A and 1B ).[20] The LacI gene
located upstream of the T7 promoter confers the inducible characteristic
of the BCD system, so the expression of recombinant proteins in
bicistronic T7 systems requires the addition of IPTG.