Fig. 2.

A construct for production of accessory genes required for [FeFe]-hydrogenase activity. (A) A construct encoding two bicistronic operons for Sh. oneidensis hydGX and hydEF was designed. The locations of promoters and engineered ribosome binding sites are shown. (B) The Sh. oneidensis hydG and hydEF genes are transcribed and translated. E. coli strain K38/pGP1-2 was transformed with plasmids: pUNI-Sh-EFG; pDuet-Sh-GX-EF; and pSU23-Sh-GX-EF and cultured in M9 minimal medium lacking cysteine and methionine and, where appropriate supplemented with 1 mM IPTG to de-repress LacI encoded on the pDuet-Sh-GX-EF plasmid. Cells were pulse-labelled with ³⁵S-methionine and protein samples were then separated by SDS–PAGE (14% w/v polyacrylamide), fixed, and visualised by autoradiography.