A C-terminal 2× repeat Flag-tag was inserted into C. jejuni fetA by FastCloning (35 (link)) using the constitutively expressing complementation vector pRRC_1651 as a template (Fig. S1K) and Q5 DNA polymerase. pRRC_1651 includes 93 C-terminal bp of fetP, 82 bp of the intergenic region, and the complete 1,404 bp of the fetA gene. C. jejuni strains ΔfetA and ΔfetABCDEF were then complemented with this construct following the method to generate ΔfetAc, producing ΔfetA2xFlag-FetA and ΔfetABCDEF2xFlag-FetA. Successful insertion was confirmed by sequencing purified genomic DNA. Methods to confirm the proper functionality of the tagged FetA variant in the deletion strains are described in the Supplemental Methods.
To examine the expression of tagged FetA in C. jejuni, the variant-complemented strains were grown to mid-log-phase in 15 mL MH-TV, resuspended in fresh MH-TV supplemented with or without 10 µM DFO, and pelleted after 3 h of growth. The harvested cell pellets were analyzed by SDS-PAGE and probed by western blot with an anti-Flag antibody (Genscript A01868).
To examine the expression of tagged FetA in C. jejuni, the variant-complemented strains were grown to mid-log-phase in 15 mL MH-TV, resuspended in fresh MH-TV supplemented with or without 10 µM DFO, and pelleted after 3 h of growth. The harvested cell pellets were analyzed by SDS-PAGE and probed by western blot with an anti-Flag antibody (Genscript A01868).
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