Table 1. Levels of PBP1ab expressed from different cassettes, and repressed using different sgRNA or crRNA.
The levels are determined using either fluorescence microscopy, SDS-PAGE with fluorescence detection, or mass spectrometry (DIA: Data-Independent Acquisition or PRM: Parallel Reaction Monitoring.), as described in the Materials and methods. RFP-PBP1a is either non-repressed (AV44) or deleted (AV51 and AV58). GFP-PBP1b is either non-repressed (AV44) or deleted (AV50 and AV63). Levels relative to LC69 are obtained by multiplying the levels relative to AV44 by the levels obtained by DIA for AV44, with propagated error. Ø: Control guides producing no repression. n.d.: not determined.
Table 1—source data 1. Data used to generate Table 1.
elife-51998-table1-data1.xlsx (20.7KB, xlsx)
| Relative and absolute quantification of PBP1b | ||||||||
|---|---|---|---|---|---|---|---|---|
| Strain | Promoter | System | Guide | Fluorescence (% of AV44) | Fluorescence (% of LC69) | Dia (%) | SDS-PAGE (copy/cell) | PRM (copy/cell) |
| LC69 | Wild-type | n.d. | n.d. | 100 | n.d. | 166 ± 28 (100%) | ||
| AV44 | Native fusion | sgRNA | G20 | 1.0 ± 0.04 | 3.8 ± 0.4 | n.d. | n.d. | n.d. |
| AV44 | Native fusion | sgRNA | G14 | 6.6 ± 0.79 | 24 ± 2.9 | 27 ± 2 | 40 ± 5 | 46 (28%) |
| AV51 | Native fusion | sgRNA | G14 | n.d. | n.d. | 33 ± 3 | 67 ± 14 | 56 ± 7 (33%) |
| AV51 | Native fusion | crRNA | G20 | 4.1 ± 2.0 | 15 ± 7.6 | n.d. | n.d. | n.d. |
| AV51 | Native fusion | crRNA | G14 | 12 ± 3.1 | 44 ± 12 | n.d. | n.d. | n.d. |
| AV51 | Native fusion | crRNA | G10 | 36 ± 2.8 | 131 ± 15 | n.d. | n.d. | n.d. |
| AV51 | Native fusion | crRNA | GØ | 97 ± 13 | 356 ± 57 | n.d. | n.d. | n.d. |
| AV44 | Native fusion | crRNA | GØ | 100 ± 5.4 | 367 ± 38 | 367 ± 32 | 688 ± 115 | 547 ± 52 (330%) |
| AV58 | Para | crRNA | GØ | 509 ± 57 | 1870 ± 265 | n.d. | n.d. | n.d. |
| Relative quantification of PBP1a | ||||||||
| Strain | Promoter | System | Guide | Fluorescence (% of AV44) | Fluorescence (% of LC69) | Dia (%) | ||
| LC69 | Wild-type | n.d. | n.d. | 100 | ||||
| AV44 | Native fusion | sgRNA | R20 | n.d. | n.d. | 20 ± 2 | ||
| AV50 | Native fusion | crRNA | R20 | 3 ± 4 | 43 ± 56 | n.d. | ||
| AV50 | Native fusion | crRNA | R18 | 21 ± 4 | 278 ± 139 | n.d. | ||
| AV50 | Native fusion | crRNA | R11 | 46 ± 6 | 620 ± 298 | n.d. | ||
| AV50 | Native fusion | crRNA | RØ | 100 ± 1 | 1337 ± 615 | n.d. | ||
| AV44 | Native fusion | crRNA | RØ | 100 ± 7 | 1337 ± 622 | 1337 ± 615 | ||
| AV63 | Para | crRNA | R18 | 166 ± 14 | 1549 ± 786 | n.d. | ||
| AV63 | Para | crRNA | R11 | 355 ± 22 | 4750 ± 2204 | n.d. | ||
| AV63 | Para | crRNA | RØ | 691 ± 50 | 9243 ± 4304 | n.d. | ||