The Staphylococcus aureus secretome - TI Pharma

Chapter 7
Materials and Methods
Bacterial strains, plasmids and growth media
The bacterial strains and plasmids used in this study are listed in Table 1.
Table 1. Strains and plasmids used in this study
Plasmids Relevant properties Reference
pDG783 pSB118 derivative; contains the kanamycin resistance marker
from Streptococcus faecalis; Amp R ; Km R
(Guérout-Fleury et
al., 1995)
pUC18 Ap R , ColE1, φ80lacZ, lac promoter (Sambrook et al.,
1989)
pX Vector for the integration of genes in the amyE locus of B.
subtilis; integrated genes will be transcribed from the xylose
inducible xylA promoter; carries the xylR gene; Amp R ; Cm R
(Kim et al., 1996)
pXTC pX derivative in which the chloramphenicol resistance marker
has been replaced with a tetracycline resistance marker; Amp R (Darmon et al.,
; 2006)
Tet R
TOPO pCR®-Blunt II-TOPO® vector; Km R Invitrogen Life
Technologies, Inc.
pMAD shuttle vector for E. coli and S. aureus with thermosensitive ori
in S. aureus; contains the bgaB gene; Em R ; Amp R
(Arnaud et al., 2004)
pMAD-mscL pMAD plasmid with the flanking regions of S. aureus mscL;
Em R ; Amp R
This work
BaSysBio II Ligation-independent cloning vector based on pDG1727 for
Promoter-GFP activity analysis; Amp R , Spec R
Stéphane Aymerich
et al., unpublished
Strains
E. coli
Genotype Reference
DH5α F - , Ф80dlacZ ∆M15 endA1 recA1 gyrA96 thi-1 hsdR17 (rk- Invitrogen Life
mk+) supE44 relA1 deoR ∆(lacZYA-argF) U169
Technologies, Inc.
TOP10 Cloning host for TOPO vector; F - mcrA ∆(mrr-hsdRMS-mcrBC)
Φ80lacZ∆M15 ∆lacX74 recA1 araD139 ∆(ara-leu)7697 galU
galK rpsL (Str R Invitrogen Life
B. subtilis
) endA1 nupG
Technologies, Inc.
168 trpC2 (Kunst et al., 1997)
168 Cm trpC2; amyE::pX; Cm R
(Dubois et al., 2009)
∆SPβ trpC2; ∆SPβ; sublancin 168 sensitive (Dorenbos et al.,
2002)
∆SPβ Tc trpC2; ∆SPβ; amyE::pXTC; sublancin 168 sensitive; Tet R
(Dubois et al., 2009)
∆mscL trpC2; pMUTIN4mcs::mscL; double crossover deletion of mscL;
Em R
BSFA collection
strain BFS1257
∆SPβ∆mscL trpC2; ∆SPβ; pMUTIN4mcs::mscL; Em R This work
Strains
B. subtilis
Genotype Reference
∆sunA-
∆yolF
trpC2; ∆yolF; ∆sunA; Km R (Dubois et al., 2009)
∆sunA trpC2; ∆sunA; Km R S. aureus
(Dubois et al., 2009)
RN4220 Restriction-deficient derivative of NCTC 8325, cured of all (Kreiswirth et al.,
known prophages; rsbU-; agr-
1983)
RN4220 RN4220 that contains the pMAD vector for erythromycin
Em resistance; Em R
This work
RN4220
∆mscL
RN4220 derivative; rsbU-; agr-; ∆mscL This work
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