2.2 Construction of RBS library
The RBS library of ThiMEc was obtained by
circular-PCR using degenerate primer including R (representing A or G)
to introduce RBS library. The primes are listed in Table S3. The linear
PCR segment with overlap region was transformed to DH5α. The
transformers covered on plate were washed with fresh Luria Bertani (LB)
and cultivated in LB medium for 12h. Then the plasmids were extracted
and transformed to BL21(DE3) with pEBI. Several transformants on agar
plates were picked and conserved, constituting the RBS library.