Contributors: Tank, Esther
Purpose
First plate combining MUC and MUG substrates, both as mixing-ratio series. Test ID 508. Gain lowered to 500 (signal was off-scale at 1000), bottom read, 2 min × 100 cycles = 198 min, 37 °C.
MUC half — clean
| Group | Rate (FSU/min) | Efficiency |
|---|---|---|
| 1:0 (rel 1.00) | 4.731 | 100% |
| 1:1 (0.50) | 2.415 | 102% |
| 1:3 (0.25) | 1.357 | 115% |
| 1:7 (0.125) | 0.017 | 3% — failed well group |
| 1:15 (0.0625) | 0.458 | 155% (small denominator) |
| 0:1 pure His | 0.040 | 0.8% of signal |
| PBS + MUC | 0.021 | — |
| BugBuster + MUC | −0.004 | — |
| 1:0 repeat (independent mix) | 4.521 | agrees with 4.731 to 4.4% |
Substrate turnover 2.8% — strictly initial-rate territory. k = 156 FSU/µM at gain 500. Scaled to culture: 3.03 mU per 5 mL lysate = 0.303 mU per mL culture = 0.109 mU per OD·mL.
MUG half — compromised
All CxnA + MUG wells plateaued at 69–72 µM equivalent across a 16-fold enzyme range, with scrambled time-to-peak. C1–3 and G7–9 were the same condition and read 11,306 vs 69. No interpretation was possible from this run (resolved on 15 September).
Problems and actions
- Gain changed mid-series → absolute FSU no longer comparable with Runs 1–4; relative results within this run unaffected.
- Export contained only “average over replicates” → no CV could be computed and no bad well could be found inside a group. Always export raw data.
- Three failed additions (A10–12, G1–3, and the MUG block) → no fix identified this round.
Cloning side, same day: codon-optimised epPCR gel, screening plates set up
Separately from the MUC/MUG work above:
Gel of epPCR on codon-optimised construct (p. 160)
0.8% agarose gel: 2 g agarose, 250 ml 1× TAE, 12.5 µl Gel Red.
Setting up Congo red / X-gluc plates
Fifteen colonies from the epPCR library (non-codon-optimised, i.e. the native-sequence library) plated out for both screens:
- Congo red: colonies 1–15
- X-gluc: colonies 1–15