Table 2. Plasmid yields and stability (see also Figs. 1 and 2). Cells were grown as overnight cultures for extraction of pUC19-based ColE1 plasmids, or pRSF or p15A non-ColE1 plasmids, expressing Cas3 or Cascade as indicated.
| E. coli strain | Plasmid | Plasmid concentration (ng/µl)a) | OD600 | Viability cells per ml (×107)c) | AmpR, KanR or ChlmR cells (% total)b) |
|---|---|---|---|---|---|
| MG1655 |
pUC19 |
68.4 (± 22.5) |
1.3 (± 0.02) |
154 (± 22) |
100.0 |
| pCas3+ |
261.3 (± 17.0) |
2.0 (± 0.07) |
123 (± 32) |
94.0 |
|
| pCas3K320L |
72.9 (± 3.4) |
2.3 (± 0.01) |
109 (± 3) |
95.5 |
|
| |
pCas3K78L |
221.8 (± 43.0) |
1.4 (± 0.17) |
78.5 (± 23) |
5.3 |
| |
pCascade |
25.6 (± 4.4) |
1.9 (± 0.13) |
173(± 7) |
0.1 |
| |
pCasC |
30.8 (± 1.3) |
1.5 (± 0.12) |
72.5(± 33) |
5.0 |
| BL21 AI |
pRSF1b |
112.7 (± 4.6) |
1.4 (± 0.05) |
70 (± 10) |
89.7 |
| |
pCas3-RSF (arabinose) |
139.0 (± 21.8) |
1.1 (± 0.12) |
32.5 (± 5) |
85.7 |
| |
pCas3-RSF (glucose) |
144.5 (± 26.0) |
1.5 (± 0.15) |
77.5 (± 20) |
100 |
| MG1655 araB T7 RNAP |
pACYCDuet-1 pCas3-pACYC (arabinose) pCas3-pACYC (glucose) |
29.5 (± 2.8) 29.5 (± 2.8) 39.75 (± 11.7) |
1.7 (± 0.01) 1.46 (± 0.04) 1.75 (± 0.05) |
115 (± 35) 65 (± 7) 115 (± 21) |
100 100 100 |
| EB304 (Δcas3) |
pCascade |
25.4(± 4.0) |
0.94 (± 0.2) |
190 (± 16) |
1.0 |
| pUC19 | 47.66(± 7.0) | 2.0 (± 0.02) | 147 (± 10) | 100.0 |
a)Plasmid concentrations were measured in a Nanovue spectrophotometer (GE Healthcare). Values are means of three independent experiments with standard deviations given in parentheses. b)The percentage cells retaining plasmid was measured by comparing viable colony counts after plating on agar with, or without ampicillin or appropriate antibiotic. c)Viable cell number was from counting colonies after plating 100 µl of serially diluted bacteria on LB agar plates from at least two independent experiments.