Nico21 de3 escherichia coli

Note that the A, B naming of the ETF subunits is opposite to their naming in the Fix convention, such that EtfA is FixB, and vice versa. Thus, the fixA gene encodes EtfB and fixB encodes EtfA. Mutations were introduced into fixB augmented with an N-terminal His tag in the pMCSG21 plasmid (spectinomycin resistant) (19 (link)). Primers encoding the desired amino acid substitutions were designed using NEB base changer (Table S2) and employed according to the vendor’s recommendations to introduce the mutations via polymerase chain reactions using Q5 High Fidelity DNA polymerase (New England Biolabs). The mutated construct encoding EtfA and plasmid encoding native EtfB were transformed into competent Nico21(DE3) Escherichia coli (New England Biolabs), along with pGro7 (Takara Bio) for coexpression of molecular chaperones GroES/EL. Plasmids expressing fixB with mutations encoding the R165H or R165K substitutions were obtained from Dr H. Diessel Duan.

Mutations were introduced into the genes for wild type RpaETF: fixA and fixB in the plasmids pMCSG28 (carbenicillin resistant) and pMCSG21 (spectinomycin resistant), respectively.3 (link) At the level of the protein, we adopt the letters S and L to identify the small and large subunit of the heterodimer, respectively, because these are homologous to the S and L subunits of other ETFs. However due to different gene orders in the fix operon the large FixAB subunit is FixB whereas the large ETF subunit is EtfA. Our proposal to adopt an ‘S’ and ‘L’ notation instead seeks to circumvent the confusion engendered by EtfAB's correspondence to FixBA. We retain the traditional notation for genes in order to retain correspondence with extensive annotation in databases.
Back-to-back orientation primers encoding the desired amino acid substitutions were designed using NEB base changer (ESI Table S1†) and employed according to the vendor's recommendations to introduce the mutations via polymerase chain reactions using Q5 High Fidelity DNA polymerase (New England Biolabs, Ipswich MA). A pair of constructs encoding EtfS and EtfL were then used to transform competent Nico21(DE3) Escherichia coli (New England Biolabs, Ipswich MA), to permit protein expression.