Dry lab & wet lab methodology

Before committing to colony-by-colony screening, we tested whether growth on cellobiose as the sole carbon source could itself serve as a selection — pool a mutant library and let only variants with real β-glucosidase-like activity grow, rather than screening colonies individually.

• Strains: E. coli TOP10 expressing wild-type CxnA, or a secreted β-glucosidase as a positive-control comparator
• Media: M9 minimal medium + chloramphenicol + 0.1% casamino acids, with 0.2% glucose or 0.2% cellobiose as the carbon source
• Conditions: inoculated at OD₆₀₀ = 0.05, grown at 37 °C, 200 rpm
• Readouts: OD₆₀₀ at 24 h; viable counts by serial dilution plating on LB + chloramphenicol

• Host strain: E. coli TOP10, used for CxnA, His-CxnA (frameshift control), and a known β-glucosidase (positive control)
• Liquid culture: standard LB medium, with or without 0.38 mM IPTG and 40 µg/mL chloramphenicol as needed, 255 rpm, 37 °C
• Plates: autoclaved LB agar microwaved 3 min to re-liquify, cooled, then 40 µg/mL chloramphenicol added before pouring into 9 cm dishes

• Kit: QIAprep Spin Miniprep Kit (Qiagen), standard centrifuge protocol
• Adaptation: where two pellets needed combining, each was resuspended in 125 µL Buffer P1 and the two suspensions transferred into the same microcentrifuge tube before continuing
• Quantification: Nanodrop, with Buffer EB as the blank

High-fidelity backbone

• Primers Start REV 2 and End FOR 2, Q5 High-Fidelity DNA Polymerase with GC Enhancer (NEB #M0491), annealing at 62 °C
• 50 µL product + 6X Purple Loading Dye (Roche), run on 0.8% agarose gel alongside a 1 kb ladder, visualised under UV
• Gel-purified with the Wizard SV Gel and PCR Clean-Up System (Promega); final elution incubation extended to 5 min

Codon-optimised insert

• Ordered as two synthetic gBlocks (OpPart1_CxnA, OpPart2_CxnA) to work around CxnA's high GC content (~71%)
• 2 ng of each gBlock + 71.5 ng Q5-amplified backbone, joined with the NEBuilder HiFi DNA Assembly Reaction Protocol

Error-prone PCR

• Taq DNA Polymerase Standard Protocol (Roche #11147633103), primers Start FOR Hx2 and End REV 1
• 5% DMSO, 0.32 mM MgCl₂, MnCl₂ gradient 0–0.1 mM to tune mutation rate, annealing at 57 °C
• Products gel-excised, quantified, then ligated to the backbone with a Gibson Assembly kit (NEB)

• Plates: LB agar + 0.2% w/v carboxymethylcellulose (CMC) + 0.38 mM IPTG + 40 µg/mL chloramphenicol, sectioned into four quadrants
• Inoculation: both colony-picking directly onto plates and spotting 1 µL of liquid culture were tested
• Incubation time: a timed 24 h experiment established that 12 h at 37 °C was sufficient before staining
• Staining: flood with 0.1% Congo Red for 15 min, destain with 1 M NaCl for 15 min, measure zones of clearance

• Substrate: 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside (X-gluc, Merck), dissolved to 40 mg/mL in 100% DMSO, then diluted 20 µL stock + 80 µL sterile water
• Plates: LB + 40 µg/mL chloramphenicol + 0.38 mM IPTG
• Inoculation: 50 µL spread directly, or 1 µL drops across four quadrants
• Development: overnight growth at 37 °C, then diluted X-gluc either spread over the whole plate or dropped (5 µL) onto individual colonies

Lysate preparation

• Overnight cultures grown with 0.38 mM IPTG from inoculation
• Harvested by centrifugation, washed once in PBS, resuspended in BugBuster lysis reagent + Roche cOmplete EDTA-free protease inhibitor
• Normalised to 5.56 OD₆₀₀·mL of culture per mL of lysis reagent, so every lysate receives a matched cell input
• Lysed 30 min at room temperature with gentle agitation, clarified by centrifugation (4000g, 5 min), held on ice; all compared lysates were prepared and assayed the same day
• Total protein estimated by A280 on Nanodrop (used only as a rough check — see the honest note on this below)

MUC reaction

• Reaction: 200 µL total = 10 µL lysate + 188 µL PBS + 2 µL MUC reagent (10 mM stock in DMSO) → 200 µM MUC in-well
• Plate: Greiner 96 F-bottom
• Reader: BMG Labtech FLUOstar Omega, bottom optic, Ex 310-10 nm / Em 460 nm, 37 °C, read every 2 min for 198 min
• Gain: 500 for the screening plate; 1000 for earlier method-development plates
• No excitation filter closer to the 4-MU anion's true absorbance maximum (≈360 nm) was available on the instrument, so all readings used 310 nm

Calibration and controls

• 4-MU standards (0–6.25 µM in-well) prepared by serial dilution in DMSO, then transferred into the assay matrix — included on every plate in both PBS and frameshift-lysate backgrounds
• Enzyme-amount proportionality established by mixing active lysate with frameshifted (CxnA-His) lysate at fixed ratios (1:0, 1:1, 1:3, 1:7, 1:15) while holding total lysate at 10 µL — a volume-varying alternative was tested first and rejected (see Results)

Colonies flagged by the Congo red, X-gluc, and MUC screens as interesting — retained activity, lost activity, or an above-baseline MUC signal — were grown in liquid culture for plasmid isolation.

• Plasmid isolation: QIAprep Spin Miniprep Kit (Qiagen)
• DNA quality check: Nanodrop 2000 (Thermo Fisher) — concentration, A260/A280, and A260/A230 ratios, before committing samples to sequencing
• Sequencing: Sanger sequencing via MRC PPU DNA Sequencing and Services