Contributors: Esther, Tank
Congo red results — picking colonies for culturing & the MUC assay (pp. 162–163)
Congo red results read (on Tank’s phone). Interesting colonies flagged for culturing and the quantitative MUC assay:
| Sample | Observation | Number |
|---|---|---|
| CxnA (control) | — | 2.25 |
| His-CxnA (control) | — | 2.41 |
| β-glucosidase (control) | — | 2.58 |
| CO 2 | clear halo, big | 2.98 |
| CO 4 | clear halo, big | 2.93 |
| CO 7 | clear halo | 3.03 |
| T 4 | big halo | 3.15 |
| T 15 | nothing | 2.79 |
| T 14 | nothing | 2.80 |
The right-hand “Number” column is unlabelled in the notebook — reproduced as written, meaning not assumed to be any particular unit or measurement. CO 12 isn’t on this list; it’s picked up the next day from the X-gluc read (see the 16 September entry).
Purpose
MUG troubleshooting plate: boiled controls, −IPTG controls, substrate and lysate dilutions, and a secretion test. Test ID 512. Gain 500, bottom read, 2 min × 100 cycles = 198 min, 37 °C, raw data exported this time.
Preceded by an endpoint read at gain 1000 in which four groups hit the 260,000 detector ceiling.
The MUG result — and what it actually means
| Group | Signal | Interpretation |
|---|---|---|
| CxnA + MUG | complete before t = 0 | — |
| CxnA-His frameshift + MUG | identical to CxnA | activity is not CxnA-dependent |
| CxnA −IPTG + MUG | also complete | not induction-dependent either |
| All three, boiled | back to background (PBS + MUG level) | heat-labile — it’s an enzyme, just not ours |
| β-glucosidase strain + MUG | background, on three separate plates | — |
| Plateau vs substrate (12.5/50/200 µM) | plateau = that amount fully hydrolysed each time | the “ceiling” seen at gain 1000 was never a detector ceiling — just the substrate supplied, fully converted |
Resolved: the substrate bottle was 4-methylumbelliferyl β-D-galactopyranoside (MUGal), not the glucopyranoside (MUG). This explains every observation: TOP10 is φ80lacZΔM15 ΔlacX74 — it supplies the ω fragment of β-galactosidase, and a plasmid-borne lacZα (present on both the CxnA and CxnA-His backbones) reconstitutes active β-galactosidase by α-complementation regardless of the CxnA insert or induction state. The dedicated β-glucosidase strain uses a different vector without this α-complementation system, so it gives no signal. X-glu (a β-glucoside) and MUGal (a β-galactoside) test different enzymes entirely — the earlier X-glu-positive / MUGal-negative-expected pattern was never a contradiction.
The MUC data are unaffected. The frameshift control sits at only 0.8% signal on MUC (Run 5), so β-galactosidase activity doesn’t touch the cellobioside substrate — the MUC assay was measuring real CxnA activity all along.
MUC read-outs from the same plate
| Measurement | Rate (FSU/min) | Note |
|---|---|---|
| CxnA +IPTG | 4.264 | — |
| CxnA −IPTG | 1.903 | promoter leakiness = 45% |
| CxnA spent medium, 10 µl | 0.605 | 14% of lysate activity |
| CxnA spent medium, 50 µl | 1.636 | 2.7× rather than 5× — matrix suppression |
| CxnA-His spent medium, 10 µl | −0.009 | zero — medium activity is CxnA-specific |
| LB blank, 10 and 50 µl | −0.014 / −0.012 | zero; blank subtraction changes nothing |
Secretion can’t be claimed from this data alone — overnight cultures always lyse to some extent, and separating true export from leakage needs a cytoplasmic marker measured in the same medium.
Problems and actions
- One MUC well was contaminated with MUG (t₀ 4,045 vs 200 in its pair) → excluded by median; the 45% leakiness figure needs a clean repeat.
- Standard curve non-monotonic again (6.25 low, 12.5 high) → the 6.25 group was probably under-loaded.
- β-glucosidase strain flat on three consecutive plates → now fully explained by the substrate identity mix-up.
Side experiment — 4-MU stock comparison
A freshly weighed 4-MU stock was compared against three older stocks, all read at a nominal 12.5 µM.
| Stock | Blank-subtracted | Duplicate spread | FSU/µM |
|---|---|---|---|
| Fresh | 102,557 | 1.6% | 8,205 |
| Old 1 | 130,884 | 11.4% | 10,471 |
| Old 2 | 131,710 | 3.9% | 10,537 |
| Old 3 (n=1) | 127,795 | — | 10,224 |
The three old stocks agree to CV 4.5% (n=5); the fresh stock reads 21.5% lower. Three independent weighings agreeing is stronger evidence than one, and 1 mL of 10 mM 4-MU requires only 1.76 mg — under-weighing by just 0.38 mg on a poor balance reproduces the discrepancy exactly. Old 2 adopted (best internal agreement). The 21% discrepancy only affects absolute µM and mU/mg values; ratios, linearity, and recovery are unchanged.
Open questions carried forward: the 45% promoter-leakiness figure needs a clean repeat, and the secretion-vs-lysis question needs a cytoplasmic marker assay.