OpenVent Cellular Reagent Production (Evaporative Drying, High-Fidelity PCR)
Produces a functional Vent DNA polymerase (Pyrococcus sp.) reagent as lyophilisation-free cellular reagent, using the same evaporative-drying method as LibreBiotech procedure 59 but with the high-fidelity OpenVent plasmid (pOBL-OpenVent) and IPTG induction in place of aTC. OpenVent provides proofreading activity suited to high-fidelity PCR, cloning, and difficult templates (GC-rich, long amplicons up to ~7.5 kb). Adapted from Bhadra et al. 2021 PLoS ONE (CC-BY 4.0). Not a clinical or regulatory-grade reagent.
Version History
Version 0.1.1 Viewing Latest
Effective: 2026-04-20All `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: 2026-04-20Initial ingestion of the OpenVent cellular-reagent workflow from Bhadra et al. 2021 PLoS ONE (CC-BY 4.0). Sibling procedure to LibreBiotech 59 (OpenTaq); differs in plasmid (pOBL-OpenVent), inducer (IPTG 1 mM), and induction duration (4 h vs. 3 h). Downstream steps reference procedures 60–64 for novice detail. Procedural facts preserved from the paper; prose written fresh; full attribution via references and EXTERNAL_URL.
Procedure Details
- PC1 (OGTR) / CL1 (HSE) containment required. Produces and manipulates a genetically modified organism (E. coli BL21(DE3) carrying pOBL-OpenVent). OGTR-certified PC1 facility (AU) or HSE-notified CL1 premises (UK) required.
- BSL-1 host. BL21(DE3) is non-pathogenic. Autoclave all liquid and solid culture waste.
- IPTG. Isopropyl β-D-1-thiogalactopyranoside is non-toxic at the concentrations used here but avoid inhalation of powder. Standard glove + eye protection when preparing stocks.
- Ampicillin. Wear gloves; autoclave antibiotic-contaminated waste before disposal.
- Heat inactivation required. 60°C × 10 min after drying, before any external distribution, to ensure no viable GMO cells remain in the product.
- RUO only. Research and educational use. Not validated or approved for clinical diagnostic use.
Plasmid + strain. Obtain pOBL-OpenVent from Addgene (Open Enzyme Collection) under OpenMTA terms. Obtain E. coli BL21(DE3) competent cells from NEB or prepare in-house. Transform per LibreBiotech procedure 60 and archive a glycerol stock per procedure 61 before beginning production.
Media prep. Superior Broth or in-house LB broth equivalent (32/20/5 g/L tryptone/yeast extract/NaCl, autoclaved). Ampicillin stock at 100 mg/mL in water, filter-sterilised, stored at −20°C.
IPTG prep. 1 M IPTG stock in water, filter-sterilised (0.22 µm), stored in 1 mL single-use aliquots at −20°C. Do not refreeze thawed aliquots — IPTG slowly oxidises in solution.
Desiccant prep. Fresh or regenerated Drierite 8 mesh calcium sulfate pellets. Regenerate by baking at 200°C for 2 h and cooling in a sealed container.
LibreBiotech setup. Before starting production: create a Process in your production Study referencing this procedure version. Lot number format: OV-YYYYMMDD-NN (OpenVent / date / batch-of-day).
Expected contrast vs. procedure 59 (OpenTaq): identical growth and harvest pipeline; only the inducer, induction duration, and plasmid change. Workflows can be run in parallel if you have two flasks and two aliquot jars.
- Day 0: plate transformation → 37°C overnight.
- Day 1: pick colony → starter culture overnight at 37°C, 225 rpm.
- Day 2 (~6 h active): 1:200 dilution into 50 mL, grow to OD600 0.4 (~2 h), induce with 1 mM IPTG, continue shaking 4 h at 37°C (vs. 3 h for Taq), harvest-wash-aliquot (~30 min).
- Day 2 → Day 3/4: evaporative drying at 37°C static incubator, 1–2 days.
- Day 4: seal, heat-inactivate (60°C × 10 min per procedure 64), activity QC, store.
- Total active time: ~7 hours across 4 days, slightly longer than Taq due to the extended induction.
-
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
Calibrated annually -
Spectrophotometer
Measurement
Specs: OD600 at 1 mL cuvette volume
Induction-timing decision -
Vortex mixer
Mixing
Specs: Standard benchtop
Resuspension steps only -
Biosafety cabinet
Safety
Specs: Class II Type A2 or equivalent; PC1 certified
OGTR/HSE compliance -
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 -
Shaking incubator
Thermal regulation
Specs: 37°C, 225 rpm, 250 mL flask positions
Growth + induction
-
E. coli BL21(DE3)
(NEB)
C2527IBacterial strain
Qty: 1 transformant
NEB C2527I; transform fresh or use glycerol stock from procedure 61 -
pOBL-OpenVent
(Addgene) Expression plasmid
Qty: 1 ng
Addgene Open Enzyme Collection; OpenMTA licensed -
Ampicillin
Antibiotic
Qty: 100 µg/mL final
pOBL-OpenVent has ampicillin resistance -
IPTG
Inducer
Qty: 1 mM final
Differs from Taq (aTC); prepare 1 M stock fresh in water, filter-sterilise, store −20°C in single-use aliquots -
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
For wash + final resuspension -
Superior Broth
(Athena Enzyme Systems)
0105Growth 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 |
|---|---|---|---|---|---|
iptg_induction_conc_mM |
number | — |
1
|
— | IPTG concentration for protein induction. 1 mM standard for T7 promoter systems. Lower concentrations reduce yield; higher wastes IPTG without proportional benefit. |
iptg_induction_time_h |
number | — |
4
|
hour (UO:0000032) | Duration of IPTG-mediated expression at 37°C, 225 rpm. 4 h per the reference paper. Shorter reduces yield; longer risks inclusion bodies. |
induction_temp_c |
number | — |
37
|
degree Celsius (UO:0000027) | Temperature during induction. 37°C standard for OpenVent (Pyrococcus-derived, thermostable). Lower temperatures reduce expression without improving folding. |
harvest_od600 |
number | — |
0.4
|
— | OD600 at which to induce. OpenVent uses a lower induction window than Taq — 0.4 (early log) vs. 0.55–0.65 (mid log). |
final_suspension_od600 |
number | — |
6.5
|
— | OD600 of concentrated suspension before aliquotting. Same as procedure 59 Taq (6.0–7.0). |
cells_per_aliquot |
number | — |
20000000
|
— | ~2 × 10⁷ cells per 3 µL aliquot. |
evaporation_temp_c |
number | — |
37
|
degree Celsius (UO:0000027) | Static incubator temperature during drying. See procedure 64. |
evaporation_time_days |
number | — |
2
|
day (UO:0000033) | Drying duration. Typically 24–48 h; longer for high-humidity conditions. |
heat_inactivation_temp_c |
number | — |
60
|
degree Celsius (UO:0000027) | Temperature for heat inactivation of GMO host. OpenVent is thermostable to >95°C, so 60°C × 10 min preserves activity fully. |
storage_temp_c |
number | — |
22
|
degree Celsius (UO:0000027) | Storage temperature for dried aliquots. Room temperature; refrigerated storage extends shelf life. |
Procedure Steps (Version 0.1.1)
Transform BL21(DE3) competent cells with 1 ng of pOBL-OpenVent plasmid DNA, using ampicillin 100 µg/mL as the selection antibiotic. Follow the full method in LibreBiotech procedure 60 (Heat-shock Transformation of Chemically Competent E. coli). 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 from the saturated overnight culture per LibreBiotech procedure 61. All future OpenVent 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, and monitor OD600 until it reaches 0.4 (typically ~2 h). Measure per LibreBiotech procedure 62. OpenVent-specific: the paper's optimal induction OD is slightly lower than OpenTaq's window (0.4 vs 0.55), so don't wait for 0.55+.
Induce expression by adding IPTG to a final concentration of 1 mM (1 µL of 1 M stock per mL culture).
Continue shaking at 37°C, 225 rpm, for 4 hours (1 h longer than the OpenTaq induction). Extended induction maximises OpenVent accumulation without substantial inclusion-body formation at 37°C.
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 one more time.
After the final wash, resuspend cells in cold 1X PBS to a final OD600 of ~6.0–7.0 (dense concentrated suspension). Confirm the reading per procedure 62.
Execute the full drying-to-storage workflow per LibreBiotech procedure 64 (Evaporative Drying in a Desiccator Jar). 3 µL aliquots into 0.2 mL 8-tube PCR strips, 18-gauge cap hole-punch, 60 mL Drierite per ~118 mL desiccator jar, evaporate 1–2 days at 37°C static, seal caps, heat-inactivate at 60°C × 10 min.
Perform activity QC on one randomly-chosen tube per batch. Rehydrate in a high-fidelity PCR master mix with a known template (e.g. 400 bp β-globin fragment or any GC-balanced 200–2000 bp target). Run standard high-fidelity cycling (98°C × 30 s / 55°C × 30 s / 72°C × 60 s × 30 cycles). Confirm band at expected size; no band in NTC. Discard entire batch if QC fails.
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 OV-YYYYMMDD-NN), Sample records per strip with storage annotations, Assay record with QC gel image attached.
Expected outcome (per 50 mL induced culture). ~8 aliquot strips (64 single-use aliquots at 3 µL each). Activity equivalent to commercial Vent or Deep Vent in high-fidelity PCR on β-globin and similar targets up to 7.5 kb. Shelf life at room temperature: at least 7 months stored in a sealed desiccator jar with fresh silica or Drierite (longer than Taq — OpenVent is exceptionally stable).
Storage. Sealed desiccator jar in a dark cabinet at 20–25°C, with fresh desiccant refreshed every 3–6 months.
Activity QC (per batch, mandatory). Rehydrate one tube in a high-fidelity PCR master mix targeting a known control template — the paper uses a 400 bp β-globin fragment, but any GC-balanced 200–2000 bp target with a validated reverse-engineered primer pair works. Include a positive control (known template) and a no-template control. Expected: clear band at predicted size, sharp and single, no band in NTC. Discard the batch if QC fails.
Comparison with commercial alternatives. OpenVent performs comparably to NEB Deep Vent or Thermo Phusion (with caveats: proofreading activity is slightly lower than Phusion; extension rate is lower). For routine cloning, colony screening, and amplifying difficult templates, it is a direct substitute.
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| Culture doesn't reach OD600 0.4 in 2 h | Antibiotic too strong; stale starter | Fresh plates; verify ampicillin stock; freshly thawed glycerol |
| Weak induction (faint PCR band) | IPTG stock degraded or not freshly prepared | Fresh IPTG aliquot; verify concentration by A260 if available |
| PCR band of expected size but smeary | Proofreading activity cleaving primers | Use matched phospho-primers or reduce reaction time |
| No band in high-fidelity PCR | OpenVent sensitive to impurities in template | Cleaner template (spin-column purification); reduce inhibitor carryover |
| Ladder of secondary bands | Extended induction yielded partially degraded enzyme | Shorter induction (3 h); verify 37°C incubator accuracy |
References
- 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
- LibreBiotech procedure 59 — OpenTaq Cellular Reagent Production (sibling procedure, shares workflow). Link protocol
- LibreBiotech procedures 60 (transformation), 61 (glycerol stock), 62 (OD600), 63 (LB agar + antibiotic plates), 64 (desiccator drying). Link protocol
- Open Bioeconomy Lab — Open Enzyme Collection. Link paper
- Addgene Open Enzyme Collection (pOBL-OpenVent and related plasmids, OpenMTA). Link paper
- OpenMTA — Open Material Transfer Agreement. Link paper