The Enterococcus faecalis Information Gate - Cellular Computing - page 113

Figure 6.3 Multiplied E. faecalis information gate.

transfer is described in Dunny et al. [5]. OG1SSp(pCF10) and OG1RF were
cultured in 1 ml of THB (Todd-Hewitt broth) at 37 ◦ C for 18 h. We added 10 µ

g
tetracycline to the culture medium of OG1SSp(pCF10) to retain the plasmid
in the host cell. After incubation, the culture soup of OG1SSp and OG1RF
was removed completely. We combined OG1SSp(pCF10) and OG1RF after
washing twice with fresh THB. The cell mixtures were resuspended in the
previously removed OG1RF soup. The cell mixtures were incubated at 37 ◦ C
for 2.5 h, and, after incubation, were spread on a THB agar plate containing
10 g/ml fusidic acid. The cells on the plates were

incubated at 37 ◦ C for 18 h. We picked a bacterial colony of OG1RF(pCF10)
and cultured it in 1 ml THB with tetracycline and fusidic acid. We added 1 ml
glycerol to the bacterial culture, which was then stocked at

µ g/ml tetracycline and 50 µ

80 ◦ C. We obtained

−

OG1RF(pAM714) in a similar manner.

Table 6.3 Strains and plasmids used in this work.

E. faecalis strain Phenotype a

OG1X Sm r

OG1RF Rf r ,Fa r

OG1SSp Sm r ,Sp r

Conjugative plasmid
pCF10 Tc r , cCF10 responsive

pAM714 Em r , cAD1 responsive

pAM351 Tc r , cPD1 responsive

pOB1 (::Tc r ) Tc r , cOB1 responsive

a Sm r ; streptmycin resistance, Rf r ; refampicin resistance, Fa r ; fusidic acid
resistance, Sp r ; spectinomycin resistance, Tc r ; tetracycline resistance,
Em r ; erythromycin resistance.