Diapositiva 1

1
Figure S1. Construction of pAL70. The Hyg
cassette was amplified from pIJ963 (Lydiate et
al. 1989) using Pfu polymerase (Stratagene) and
oligonucleotides RP594 and RP595 (Fig. S5)
carrying the dif sequence of M. tuberculosis and
a BglII restriction site at their 5 ends. The
resulting fragment was cloned into the single
BclI site present in the mycobacterial
integrative vector pSM316 (unpublished).
2
Figure S2. Construction of pAL74 and pAL75. A)
The Hyg cassette was amplified from pIJ963
(Lydiate et al. 1989) using Pfu polymerase
(Stratagene) and oligonucleotides RP594 and RP803
(Fig. S5) carrying the dif sequence of M.
tuberculosis and a BglII restriction site at
their 5 ends. RP803 was designed to introduce a
StuI restriction site immediately before the dif
sequence at the 3 of the cassette. This fragment
was cloned into in pCR-BluntII-TOPO (Invitrogen)
to obtain pAL71 (A). The mycobacterial promoters
Pmpt64 and PRv1818c were amplified from pMV2-33
(Delogu et al. 2004a) and pMV4-36 (Delogu et al.
2004b), respectively using Pfu polymerase using
oligonucleotides RP804/805 and RP809/805 (Fig.
S5) containing a DraI restriction site on their
5 ends and designed to include the promoter
original rbs into the construct (B) these two
fragments were cloned into the single StuI site
of pAL71 to obtain pAL74 and pAL75 (C).
3
P
dif
dif
Hyg
Integration
P
Hyg
Xer-cise
Figure S3. Schematic representation of the effect
of integration and excision of the Hyg excisable
cassette on transcription and translation of the
downstream gene(s). Disruption of the gene of
interest (orange) by single-step double crossover
does not interrupt transcription of the
downstream gene (green) due to the promoter
present at the 3 of the Hyg excisable cassette.
After excision of the cassette, transcription
from the physiological promoter is resumed. Even
if the two genes are translationally coupled the
translation of the downstream gene(s) is not
interrupted due to the translation of a truncated
form of the gene of interest also in the presence
of the Hyg cassette. mRNA is shown as broken line.
4
Figure S4. Constructin of pDario1 and pDario2.
Both vectors are derivative of the integrative
plasmid pMV306 (Stover et al. 1991). The
hygromycin cassettes from pAL74 and pAL75,
extracted by HindIII/XbaI digestion and the GFP
coding sequence, extracted from pMV10-25 (Delogu
et al. 2004b) by NheI/KpnI digestion, were
inserted into pMV306 digested HindIII/KpnI.
5
Figure S5. Fluorescent microscopy (50X) of M.
smegmatis strains SM140 (A), SM141 (B), SM143
(C), and SM144 (D).
6
Table S1. Oligonucleotides used in this study
Sequencea
Name
BglII
RP594 5-AGATCT T AAG CCG ATA AGC GAC ATT ATG
TCA AGT CTG CAG GTC GTC GAG GTC-3 RP595
5-AGATCT A CTT GAC ATA ATG TCG CTT ATC GGC TTA
GGA TGC CAG GGC CTT TC-3
RP803 5-AGATCT A CTT GAC ATA ATG TCG CTT ATC
GGC TTA AGGCCT GGA TGC CAG GGC CTT TC-3
DraI
StuI
RP804 5-TTTAAA TCG AGC ACC ACG CGA CAC-3
RP805 5-TTTAAA ATC TCG CCC TTG CTC ACC AT-3
RP809 5-TTTAAA CTT GCC GGG ACG AGC TTC-3
MluI
RP818 5-ACGCGT GCT ACC ACT ACC GCC ACC CG-3
BglII
RP819 5-AGATCT ATG GTG GCC CAG AAC AGT TCG G-3
BglII
RP820 5-AGATCT CAC AGC GAC GGC AGC GAA GC-3
BamHI
RP821 5-GGATCC GAC GCA GAT GTT CCA GCC CG-3
MfeI
RP893 5-AAA CAATTG CCG CAG CTG CTC GAC GCT
CT-3
BglII
RP894 5-AAA AGATCT TGG TCC GTG CGG GCG TTG-3
BglII
RP895 5-AAA AGATCT GTC ATC CCG CAG GTG TCC AT
-3
SpeI
RP896 5-AAA ACTAGT GTA CTC GGT GGC CTT GGC AA
-3
aPutative dif sequences are boxed restriction
sites are underlined.
7
References

• Lydiate, D. J., A. M. Ashby, D. J. Henderson, H.
  M. Kieser, and D. A. Hopwood. 1989. Physical and
  genetic characterization of chromosomal copies of
  the Streptomyces coelicolor mini-circle. Journal
  of General Microbiology. 135941-955.
• Delogu, G., A. Bua, C. Pusceddu, M. Parra, G.
  Fadda, M. J. Brennan, and S. Zanetti. 2004a.
  Expression and purification of recombinant
  methylated HBHA iin Mycobacterium smegmatis. FEMS
  Microbiol Lett. 23933-39.
• Delogu, G., C. Pusceddu, A. Bua, G. Fadda, M. J.
  Brennan, and S. Zanetti. 2004b. Rv1818c-encoded
  PE_PGRS protein of Mycobacterium tuberculosis is
  surface exposed and influences bacterial cell
  structure. Mol Microbiol. 52725-733.
• Stover, C. K., V. F. de la Cruz, T. R. Fuerst, J.
  E. Burlein, L. A. Benson, L. T. Bennett, G. P.
  Bansal, J. F. Young, M. H. Lee, G. F. Hatfull,
  and et al. 1991. New use of BCG for recombinant
  vaccines. Nature. 351456-460.