(iii) Construction of complementation plasmids
  1. pWY54 (Ptet-mbcS+ ). A 1648-bp fragment containing the mbcS Shine Delgarno sequence and coding sequence was PCR-amplified from the USA300 LAC* chromosome using primer pair oWY470/oWY471. This also appends the sequence (DYKDDDDK) for a C-terminal FLAG epitope tag (Hopp et al., 1988). pWY53 as a backbone plasmid was amplified with primer pair oWY472/oWY473. The DNA fragments were assembled by the Gibson method and the DNA mixture was transformed into E. coli DH5α, resulting in pWY54. This places the production of Sa MbcS-FLAG under the control of the inducible Ptet promoter by anhydrotetracycline addition.
  2. pWY55 (Ptet-mbcS [K510A]). The 1523-bp fragment containing the mbcS Shine Delgarno sequence (25bp) and coding sequence (1498bp) was PCR-amplified from the USA300 LAC* chromosome using the primer pair oWY470 and oWY500. A point mutation in the active site (K510A) of mbcS was generated by a gblock (125bp, oWY499). As before, pWY53 as a backbone plasmid was amplified with primer pair oWY469 and oWY473. The DNA fragments were assembled by the Gibson method and the DNA mixture was transformed into E. coli DH5α, resulting in pWY55. This results in the production ofSa MbcSK510A-FLAG under the control of the inducible Ptet promoter by anhydrotetracycline addition.
  3. pWY75 (Ptet-RpibuA+ ). A 1716-bp fragment containing the coding sequence of RpibuA + and a FLAG tag at the C-terminus fromR. palustris was optimized and synthesized by GenScript and the RBS and ORF from SambcS were utilized. This fragment was inserted into plasmid pWY53 and verified by GenScript. The resulting plasmid was transformed into E. coli DH5α, resulting in pWY75. The plasmid directs the synthesis of Rp IbuA-FLAG under the control of the inducible Ptet promoter by anhydrotetracycline addition.
(iv) Construction of plasmids used for protein overproductio n
pFM01. A 1636-bp fragment containing theSambcS+ coding sequence was amplified fromS. aureus LAC using oWY543 and oWY544. In parallel, pTEV5 (Rocco et al., 2008) was amplified from plasmid pRpIBUA1 using oWY545 and oWY546. Both DNA fragments were assembled via the Gibson method (Gibson et al., 2009) and the product was transformed into E. coli DH5α, resulting in pFM01.
pFM03. The SambcS allele coding for theSa MbcSK510A variant protein was amplified from pWY55 using oWY543 and oWY544. In parallel, pTEV5 was amplified using oWY545 and oWY546. The DNA fragments were assembled via the Gibson method (Gibson et al., 2009) and the mixture was transformed into E. coli DH5α, resulting in pFM03.