B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

1989). 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) and
isopropyl-β-D-thiogalactopyranoside (IPTG) were from Diagnostic Chemicals Ltd.
39
(Charlottetown, Prince Edward Island, Canada). Restriction enzymes and T4 DNA ligase
were from New England Biolabs (Beverly, MA). Other chemicals were from Fisher
Scientific (Norcross, GA).
Bacterial Strains and Growth Media
Bacterial strains used in this study are listed in Table 2-1. The minimal medium
used was NCE (Vogel and Bonner 1956, Berkowitz et al. 1968) supplemented with 0.4%
1,2-propanediol, 200 ng/ml of CNCbl, 1 mM MgSO4, 0.3 mM each of valine, isoleucine,
leucine, and threonine. LB medium was the rich medium used (Difco Laboratories,
Detroit, MI) (Miller 1972). MacConkey and aldehyde indicator media were
supplemented with 1,2-propanediol and CNCbl and prepared as previously described
(Johnson et al. 2001).
General Protein and Molecular Methods and ATR Assays
Bacterial transformation, polymerase chain reaction (PCR), restriction enzyme
digests, and other standard molecular and protein methods were performed as previously
described (Maniatis et al. 1982, Johnson et al. 2001). ATR assays were also performed
as previously reported (Johnson et al. 2001).
P22 Transductions
Transductional crosses were performed as described previously (Davis et al. 1980).
In preparing lysates of galE mutant strains, overnight cultures were grown on LB
medium supplemented with 0.2% galactose and 0.2% glucose with the addition of
appropriate antibiotics. A P22 HT105/1 int-201 phage was used for transductional
crosses at a concentration of 2x10 8 phage/ml (Schmieger 1971).