Contributors: Esther, Tank
Purpose
Quantify exoglucanase activity for the seven variants flagged by the Congo red and X-gluc screens, each against a wild type grown and lysed alongside it on the same day. Test ID 513, started 14:07.
Greiner 96 F-bottom plate read on the BMG Labtech FLUOstar Omega through the bottom optic: excitation 310 nm (10 nm bandpass), emission 460 nm, gain 500, 37 °C, 100 cycles at 120 s (198 min), 20 flashes per well per cycle, double-orbital shaking at 300 rpm for 2 s before each cycle, 0.5 s settling.
Twenty-eight groups of three wells. Rows A–C carried the fresh preparation, rows D–E a stored preparation of the same strains, E10–G3 the blanks and 4-MU standards, and G4–G9 repeat wells of WT-CO colony 1 and WT-T.
Result interpretation
Rates are blank-corrected medians of three wells. Codon-optimised strains are divided by WT-CO (mean of two colonies), native-sequence strains by WT-T.
| Group | Wells | Median rate (FSU/min) | vs parent |
|---|---|---|---|
| WT-CO colony 1 | A1–A3 | 0.438 | reference |
| WT-CO colony 2 | A4–A6 | 0.523 | reference |
| WT-T | B10–B12 | 0.490 | within 1.1% of WT-CO |
| CO12 colony a | A7–A9 | 0.669 | 1.39× |
| CO12 colony b | A10–A12 | 0.627 | 1.31× |
| CO2 | B1–B3 | 0.346 | 0.72× |
| CO4 | B4–B6 | 0.473 | 0.99× |
| CO7 | B7–B9 | 0.272 | 0.57× |
| T4 | C1–C3 | 0.075 | 0.16× |
| T14 | C4–C6 | −0.011 | −0.02× |
| T15 | C7–C9 | −0.008 | −0.02× |
| CxnA-His frameshift | C10–C12 | −0.018 | no activity |
The two wild types agreed to within 1.1%, so codon optimisation altered neither expression nor catalysis. CO12 is the only variant above its parent. T14 and T15 are indistinguishable from the frameshift control.
Secondary readouts
| Measurement | Wells | Rate (FSU/min) |
|---|---|---|
| PBS + MUC blank | E10–E12 | 0.0000 (defines zero) |
| Buffer, no MUC | F1–F3 | 0.0162 |
| 4-MU standard, 0 µM | F4–F6 | 0.0130 |
| 4-MU standard, 1.5625 µM | F7–F9 | −0.0250 |
| 4-MU standard, 3.125 µM | F10–F12 | −0.0744 |
| 4-MU standard, 6.25 µM | G1–G3 | −0.1511 |
The standards decline over the read, roughly in proportion to concentration (about −0.024 FSU/min per µM at 3.125 and 6.25 µM). This is the optical loss characterised separately, not substrate turnover — the standards contain no enzyme.
Triplicate wells agreed to a median CV of 5.1% across groups. Independently grown colonies of one genotype — two each of WT-CO and CO12 — differed by 9.5%, giving a 3σ detection threshold of 1.40× for one colony measured against one wild-type colony.
Problems and actions
- One aberrant well in four of twelve fresh groups: CO12 colony a well 3 read 0.912 against 0.665 and 0.658; CO4 well 3 read 0.271 against 0.475 and 0.468; WT-T well 2 read 0.349 against 0.499 and 0.485; T4 well 1 read 0.013 against 0.070 and 0.076. Group values are therefore reported as medians rather than means.
- The stored pellets of CO7 and T4 had settled before the lysate was loaded. Stored CO7 read 0.836 FSU/min, 2.1× its stored parent, against 0.57× in the fresh preparation; stored T4 read 0.135, or 0.34×, against 0.16× fresh. A₂₈₀ was 0.7 at the meniscus versus 3–4 after mixing. Both stored values excluded.
- No WT-CO was included in the stored preparation, so stored codon-optimised strains could only be divided by the stored WT-T.
Open questions carried forward
- CO12 sits at the detection threshold at 1.39× and 1.31×. Three colonies per genotype would be needed to resolve a difference of this size.
- The 9.5% colony CV rests on two colony pairs within a single preparation. Whether it represents the variation that actually limits between-strain comparisons is untested.