(iii) Construction of complementation plasmids
- 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.
- 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.
- 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.