Contributors: Esther, Chloe, Alexander
Beta-glucosidase plasmid transformation (positive control)
Transformed competent E. coli with beta-glucosidase plasmid as a positive control for cellobiose selection. Nanodropped DNA first to confirm concentration. Standard heat-shock protocol.
Plated on chloramphenicol (His-tagged and non-tagged CxnA), growth observed ×2 each.
Native CxnA activity tests
Set up CenA on CMC (Congo red assay) and Cex on MUC plates to test native enzyme activity.
Control plate results (conditions A, B, C, D) — 1 overnight incubation
| Condition | Dilution | Result |
|---|---|---|
| (a) LB + chloramphenicol | All dilutions | TMC (too many to count) |
| (d) M9 + glucose + chloramphenicol | Undiluted | TMC / lawn-like growth |
| (d) M9 + glucose + chloramphenicol | 1:100, 1:1000 | No growth |
| (b) M9 + cellobiose + glucose | All | No growth |
| (c) M9 + cellobiose only | All | No growth |
CxnA on chloramphenicol + IPTG: undiluted = TMC; 1:100 = no growth.
Interpretation: WT CxnA shows no growth on cellobiose-only plates, consistent with the expectation that it lacks beta-glucosidase activity. Glucose plates show growth as expected (cells can use glucose regardless of CxnA activity).
Miniprep #2
Miniprepped CxnA and CxnA-His (#2), same protocol as before.
| Sample | Concentration (ng/µl) | A260/A280 | A260/A230 |
|---|---|---|---|
| CxnA-His M2 | 133.29 | 1.874 | 2.174 |
| CxnA M2 | 111.88 | 1.879 | 2.187 |