CC1(C)S[C@@H]2[C@H](NC([C@H](N)C3C=CC=CC=3)=O)C(=O)N2[C@H]1C(O)=O.C[C@H]1O[C@H]2O[C@H]3[C@H](O[C@@]2(O)C(=O)C1)[C@@H](NC)[C@@H](O)[C@@H](NC)[C@@H]3O.CC(NC)C1OC(OC2C(O)C(OC3OCC(O)(C)C(NC)C3O)C(N)CC2N)C(N)CC1.[O:81]=[CH:82][C@@H:83]([C@H:85]([C@H:87]([C@@H:89]([CH2:91][OH:92])[OH:90])[OH:88])[OH:86])[OH:84]>OCC(CO)O>[OH:81][CH:82]1[O:90][C@H:89]([CH2:91][OH:92])[C@H:87]([OH:88])[C@H:85]([OH:86])[C@H:83]1[OH:84]Eight co-integrants each from the crosses summarized in Table 2 were streaked for single colonies (from cultures cryo-preserved in 10% glycerol; described in Section 6.4)) on Luria broth plates with ampicillin (100 μg/ml), spectinomycin (50 μg/ml) and gentamycin (10 μg/ml). Single colonies were inoculated into Luria broth liquid (without drug) and incubated overnight at 37° C. with gyratory shaking. Ten microliters (each) from these cultures were spread on a MacConkey Agar (base; Becton Dickson, Difco Division, Franklin Lakes, N.J.) plated with 2% galactose and 100 μg/ml ampicillin. Following overnight growth three types of galactose-resistant colonies appeared on the agar surface: red colonies (with various morphologies), white opaque colonies and white translucent colonies, in numbers varying from a few dozen to several hundred. Resolved co-integrants were among the white translucent colonies, and a single white colony was picked from each spot, and re-streaked for purification on the same MacConkey agar. These purified white colonies were tested for spectinomycin resistance (50 μg/ml) and gentamycin resistance (10 μg/ml). The resultant colony types are summarized below in Table 4 (Gent=gentamycin, Spec=spectinomycin, R=resistance, and S=sensitivity)
読み込み中…
