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Bst-LF Cellular Reagent Production (LAMP Isothermal Amplification)

Produces a functional Bst polymerase Large Fragment reagent (Bacillus stearothermophilus) as cellular reagent without purification. Bst-LF is a strand-displacing DNA polymerase that enables loop-mediated isothermal amplification (LAMP) — field-deployable pathogen detection and biosensing without a thermocycler. Uses the same aTC-inducible pATetO-family vector as procedure 59 (OpenTaq) with identical induction conditions, but the activity QC is a LAMP reaction rather than standard PCR. Adapted from Bhadra et al. 2021 PLoS ONE (CC-BY 4.0). Not a clinical or regulatory-grade reagent.

sample_prep https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0252507
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Version History
Version 0.1.1 Viewing Latest
Effective: 2026-04-20

All `procedure N` references converted to Markdown hyperlinks pointing at https://librebiotech.org/?action=show&id=N — enables in-app click-through to referenced sibling procedures. Text content otherwise preserved.
Version 0.1.0
Effective: 2026-04-20

Initial ingestion of the Bst-LF cellular-reagent workflow from Bhadra et al. 2021 PLoS ONE (CC-BY 4.0). Production pipeline identical to procedure 59 (OpenTaq) — same aTC induction, same drying method — with LAMP-specific activity QC. Content written fresh; full attribution via references and EXTERNAL_URL. Unlocks downstream LAMP-based content arcs.

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Procedure Details
Safety & Hazards
  • PC1 (OGTR) / CL1 (HSE) containment required. E. coli BL21(DE3) carrying the Bst-LF expression plasmid is a GMO. OGTR-certified PC1 facility (AU) or HSE-notified CL1 premises (UK) required.
  • BSL-1 host. Non-pathogenic. Autoclave all liquid and solid culture waste.
  • aTC handling. Anhydrotetracycline is light-sensitive. Prepare stocks in amber tubes; store −20°C; avoid prolonged light exposure.
  • Ampicillin handling. Wear gloves; autoclave antibiotic-contaminated waste.
  • Heat inactivation required. 60°C × 10 min after drying. Bst-LF is less thermostable than Taq but fully survives this treatment (operating temperature range 60–72°C).
  • LAMP QC precautions. LAMP is exceptionally sensitive; aerosolised amplicons from prior reactions are the main failure mode. Dedicated pre-LAMP / post-LAMP workflow areas with separate pipettes. Bleach surfaces between batches.
  • RUO only. Research and educational use. Not validated for clinical diagnosis.
Preparation Notes

Plasmid + strain. Obtain the Bst-LF expression plasmid from the Open Enzyme Collection on Addgene (search for 'Bst' or 'pATetO-BstLF'; exact Addgene ID varies per depositor). Compatible alternatives include any pATetO-based Bst-LF construct. Transform BL21(DE3) per procedure 60, archive glycerol stock per procedure 61, before production.

Media prep. Superior Broth + ampicillin 100 µg/mL. Pre-warmed to 37°C before inoculation.

aTC prep. 1 mg/mL anhydrotetracycline stock in 50% ethanol, stored at −20°C in amber tubes, protected from light. Single-use aliquots recommended — repeated thawing oxidises aTC.

Desiccant prep. Fresh or regenerated Drierite 8 mesh. Regenerate at 200°C × 2 h; cool in sealed container before reuse.

LAMP QC prep. Prepare LAMP primers (F3, B3, FIP, BIP for standard LAMP; add loop primers LF and LB for accelerated LAMP) targeting a known control sequence. dNTPs, MgSO₄, and a fluorescent intercalator (SYBR Green I, EvaGreen) or OSD probe for readout. A heat block at 65°C for the activity assay.

LibreBiotech setup. Create Process in production Study. Lot number format: BL-YYYYMMDD-NN (Bst-LF / date / batch-of-day).

Expected contrast vs. procedure 59 (OpenTaq): identical induction conditions; identical drying workflow; only the plasmid and the activity QC differ. The procedures can be run in parallel on the same bench day with shared equipment.

Timing
  • Day 0: plate transformation → 37°C overnight.
  • Day 1: pick colony → starter culture overnight at 37°C, 225 rpm.
  • Day 2 (~5 h active): 1:200 dilution → grow to OD600 0.4–0.7 (~2 h) → induce with 20 ng/mL aTC (3 h at 37°C) → harvest-wash-aliquot (~30 min).
  • Day 2 → Day 3/4: evaporative drying at 37°C, 1–2 days.
  • Day 4: seal, heat-inactivate (60°C × 10 min), LAMP activity QC, store.
  • Total active time: ~6 hours across 4 days (same as Taq).
Equipment (Catalog) 9
  • Microcentrifuge Centrifugation
    Specs: ≥9,000 × g, 1.5 mL and 50 mL adapters
    Harvest + wash
  • Micropipette Liquid handling
    Specs: P10, P200, P1000; filter tips only
    Dedicated pre-LAMP / post-LAMP sets ideally
  • Spectrophotometer Measurement
    Specs: OD600 at 1 mL cuvette volume
  • Vortex mixer Mixing
    Specs: Standard benchtop
  • Biosafety cabinet Safety
    Specs: Class II Type A2 or equivalent; PC1 certified
    All open-culture steps
  • Desiccator jar Storage
    Specs: ~118 mL sealed plastic or glass jar
    Reusable; clean between batches
  • Benchtop incubator Thermal regulation
    Specs: 37°C static, ≥5 L chamber
    Evaporation phase
  • Heat block Thermal regulation
    Specs: 65°C capable, ±1°C
    For LAMP activity QC; also heat-inactivation
  • Shaking incubator Thermal regulation
    Specs: 37°C, 225 rpm, 250 mL flask positions
    Growth + induction
Materials (Catalog) 8
  • E. coli BL21(DE3) (NEB) C2527I Bacterial strain
    Qty: 1 transformant
    NEB C2527I or equivalent; Acella strains (endA-/recA-) also used in the paper
  • Expression plasmid Biological material
    Qty: 1 ng
    From Open Enzyme Collection on Addgene; verify Addgene ID per your source. Any pATetO-based Bst-LF construct works
  • Ampicillin Antibiotic
    Qty: 100 µg/mL final
    pATetO vector family typically uses ampicillin selection
  • Anhydrotetracycline Inducer
    Qty: 20 ng/mL final
    Same as OpenTaq; light-sensitive, amber tube, −20°C
  • Drierite 8 mesh (W. A. Hammond Drierite) Desiccant
    Qty: 60 mL per jar
    Regenerable at 200°C × 2 h
  • 0.2 mL 8-tube PCR strip PCR tube strip
    Qty: 2-3 strips per jar
    Individual caps required
  • Buffer Reagent
    Qty: 2 mL
    Wash + resuspension
  • Superior Broth (Athena Enzyme Systems) 0105 Growth media
    Qty: 50 mL
    LB broth equivalent acceptable
Protocol Parameters Captured per-assay on each run; exported as ISA-Tab Parameter Value columns
Name Type Required Default Unit Description
atc_induction_conc_ng_ml number — 20 — aTC concentration for induction. 20 ng/mL standard — same as OpenTaq procedure.
atc_induction_time_h number — 3 hour (UO:0000032) Duration of aTC induction at 37°C, 225 rpm. 3 h per paper.
induction_temp_c number — 37 degree Celsius (UO:0000027) Induction temperature. 37°C standard.
harvest_od600 number — 0.55 — OD600 at which to induce. Paper specifies 0.4–0.7 range; default midpoint.
final_suspension_od600 number — 6.5 — OD600 of concentrated suspension before aliquotting.
lamp_qc_temp_c number — 65 degree Celsius (UO:0000027) Incubation temperature for LAMP activity QC. 65°C is within Bst-LF's optimal range.
lamp_qc_time_min number — 45 minute (UO:0000031) LAMP QC incubation duration. 30–60 min typical; shorter if strong positive signal visible earlier.
heat_inactivation_temp_c number — 60 degree Celsius (UO:0000027) Heat inactivation of GMO host. 60°C × 10 min inactivates E. coli; Bst-LF's operating range extends to 72°C, so activity is preserved.
heat_inactivation_time_min number — 10 minute (UO:0000031) Duration of heat inactivation. 10 min standard; longer does not improve GMO elimination but risks progressive enzyme loss.
storage_temp_c number — 22 degree Celsius (UO:0000027) Storage temperature for dried aliquots. 20–25°C standard; refrigerated extends shelf life.
Procedure Steps (Version 0.1.1)

Transform BL21(DE3) competent cells with 1 ng of Bst-LF expression plasmid (pATetO-BstLF or equivalent from Open Enzyme Collection), using ampicillin 100 µg/mL as the selection antibiotic. Follow LibreBiotech procedure 60 (Heat-shock Transformation). Plate onto LB + ampicillin agar prepared per procedure 63.

Incubate transformation plates inverted at 37°C for 14–16 hours. Expect well-isolated colonies.

Pick one well-isolated colony into 3–5 mL Superior Broth + 100 µg/mL ampicillin. Grow overnight at 37°C, 225 rpm.

Prepare a glycerol stock per LibreBiotech procedure 61. All future Bst-LF batches start from this stock.

Dilute the overnight culture 1:200 into 50 mL Superior Broth + 100 µg/mL ampicillin in a 250 mL flask.

Incubate at 37°C, 225 rpm, until OD600 reaches 0.4–0.7 (~2 h). Measure per LibreBiotech procedure 62.

Induce expression by adding anhydrotetracycline to 20 ng/mL final concentration (identical to OpenTaq induction — same vector family).

Continue shaking at 37°C, 225 rpm, for 3 hours.

Harvest cells by centrifugation at 9,000 × g for 1 minute at room temperature. Discard supernatant.

Resuspend pellet in 1 mL cold 1X PBS. Centrifuge again at 9,000 × g for 1 minute. Discard supernatant. Repeat wash once more.

After the final wash, resuspend cells in cold 1X PBS to a final OD600 of ~6.0–7.0. Confirm reading per procedure 62.

Execute the full drying-to-storage workflow per LibreBiotech procedure 64. 3 µL aliquots, 60 mL Drierite per jar, 37°C static drying 1–2 days, seal, heat-inactivate at 60°C × 10 min. Note: Bst-LF has an operating range of 60–72°C, so 60°C × 10 min both inactivates the GMO host and falls within Bst-LF's activity window — enzyme remains functional.

Perform LAMP activity QC on one randomly-chosen tube per batch. Rehydrate in a LAMP master mix with a validated control primer set and template. Incubate at 65°C for 30–60 min. Confirm amplification in positive control (fluorescence rise, turbidity, or pH-indicator colour change); confirm no amplification in NTC. Discard batch if QC fails or NTC contaminates.

Store QC-passed strips in a sealed desiccator jar with fresh Drierite in a dark cabinet at 20–25°C. Record the batch in LibreBiotech: Process record with procedure_version_id, lot number (format BL-YYYYMMDD-NN), Sample records per strip, Assay record with QC LAMP fluorescence trace or gel attached.

Completion Notes

Expected outcome (per 50 mL induced culture). ~8 aliquot strips (64 single-use aliquots). Activity comparable to commercial Bst 2.0 in LAMP reactions on target templates. Shelf life at room temperature: at least 2 months post-evaporation (verified by the reference paper); refrigerated storage extends further.

Storage. Sealed desiccator jar with fresh Drierite, dark cabinet, 20–25°C. Working stocks can be kept at room temperature; archive stocks at 4°C for longer-term use.

Activity QC (per batch, mandatory). Rehydrate one tube in a LAMP master mix targeting a validated control amplicon (e.g. E. coli malB gene or any well-characterised LAMP target). Incubate at 65°C in a heat block for 30–60 minutes with periodic fluorescence monitoring (if equipped) or endpoint visual turbidity / colour-change readout. Expected: positive amplification in the control tube (fluorescence increase, turbidity, or pH-indicator colour shift) with no amplification in a no-template control. Discard batch if QC fails.

Functional notes.

  • Bst-LF reactions are isothermal: no thermocycler required — any device that maintains 60–65°C for 30 minutes works (heat block, water bath, DIY heated chamber).
  • Cross-contamination risk is higher for LAMP than for PCR because LAMP generates vast amounts of amplicon; strict pre- and post-LAMP workflow separation is mandatory.
  • Bst-LF is strand-displacing but not proofreading; not suitable for high-fidelity work (use procedure 65 OpenVent for that).

Troubleshooting.

Symptom Likely cause Fix
QC LAMP shows amplification in NTC Workflow cross-contamination Dedicated pre-LAMP area; fresh pipettes; bleach all surfaces
Slow / weak LAMP amplification on positive control Insufficient Bst-LF activity Retest induction (fresh aTC stock); verify 3 h induction duration
Reaction works at high template concentration but not dilute Batch-to-batch inconsistency Include a standard curve in every QC batch; accept only batches matching reference activity
Tubes don't dry completely in 48 h Desiccant saturated Regenerate desiccant; reduce ambient humidity
Fluorescence plateau at low level Insufficient enzyme per tube Verify cell-suspension OD600 at aliquot time; increase aliquot volume in v0.1.1 if systemic
References
  1. Bhadra S, Nguyen V, Torres J-A, Kar S, Fadanka S, Gandini C, Akligoh H, Paik I, Maranhao AC, Molloy J, Ellington AD (2021). Producing molecular biology reagents without purification. PLoS ONE 16(6):e0252507. DOI Link paper
  2. Maranhao AC, Bhadra S, Paik I, Walker D, Ellington AD (2020). An improved and readily available version of Bst DNA Polymerase for LAMP, and applications to COVID-19 diagnostics. PLoS ONE 15(12):e0242704. (Br512 variant background) DOI paper
  3. Notomi T et al. (2000). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28(12):E63. (Foundational LAMP method paper) DOI paper
  4. LibreBiotech procedures 60–64 (transformation, glycerol stock, OD600, LB plates, desiccator drying). Link protocol
  5. LibreBiotech procedure 59 — OpenTaq Cellular Reagent Production (sibling workflow). Link protocol
  6. Open Bioeconomy Lab — Open Enzyme Collection (Bst-LF plasmid source). Link paper