RTX Reverse Transcriptase Cellular Reagent Production (One-Enzyme RT-PCR)
Produces a functional RTX reverse transcriptase reagent as cellular reagent without purification. RTX is an engineered polymerase with both reverse-transcriptase and DNA-polymerase activity (Ellefson et al. 2016 Science) — enables one-tube, one-enzyme RT-PCR and RT-qPCR, including viral diagnostic assays like SARS-CoV-2 N1 and eDNA RT-qPCR. Uses IPTG induction at low temperature (18°C overnight) rather than aTC at 37°C, and kanamycin-resistant pET-28a-family vector. 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 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 Viewing
Effective: 2026-04-20Initial ingestion of the RTX cellular-reagent workflow from Bhadra et al. 2021 PLoS ONE (CC-BY 4.0), with background from Ellefson et al. 2016 Science on RTX engineering. Differs from procedure 59 (OpenTaq) in induction system (IPTG overnight at 18°C vs aTC 3 h at 37°C) and selection (kanamycin pET-28a vs ampicillin pATetO). Content fresh; full attribution preserved. Unlocks one-enzyme RT-PCR and RT-qPCR arcs.
Procedure Details
- PC1 (OGTR) / CL1 (HSE) containment required. E. coli BL21(DE3) carrying the RTX expression plasmid is a GMO.
- BSL-1 host. Non-pathogenic; autoclave all waste.
- Kanamycin handling. Kanamycin is less allergenic than ampicillin but still requires gloves. Autoclave antibiotic waste.
- IPTG handling. Non-toxic at use concentrations; handle with standard PPE; avoid powder inhalation when preparing stocks.
- 18°C shaking incubator. RTX induction requires an incubator capable of cooling below room temperature. Set ahead of time so the chamber is stable at 18°C before transfer.
- Heat inactivation required. 60°C × 10 min after drying.
- RT-qPCR QC precautions. RT-qPCR uses fluorescent intercalators (EvaGreen, SYBR) that stain DNA. Standard DNA-stain precautions — gloves, no skin contact, sealed waste.
- RUO only. Research and educational use. The paper's SARS-CoV-2 detection context does NOT constitute clinical validation for this cellular-reagent form — any clinical-decision use is strictly off-label.
Plasmid + strain. Obtain an RTX expression plasmid (pET-28a-based T7 construct with RTX insert) from the Ellington lab via Addgene, or request from OBL. Transform BL21(DE3) per procedure 60 and archive glycerol stock per procedure 61 before production.
Media prep. Superior Broth + kanamycin 30 µg/mL (standard for pET-28a). Pre-warmed to 37°C for inoculation.
IPTG prep. 1 M IPTG stock in water, filter-sterilised, stored −20°C in single-use aliquots.
18°C shaking incubator prep. Set the shaking incubator to 18°C at least 1 h before use — chambers take time to equilibrate. Some incubators cannot reach below ambient without active cooling; verify your unit is capable.
Desiccant prep. Fresh or regenerated Drierite 8 mesh. Regenerate at 200°C × 2 h.
RT-qPCR QC prep. Validated control RNA template (e.g. a synthetic in vitro transcript, or a known RT-qPCR positive-control pool). RT-qPCR primers specific to the control. EvaGreen or SYBR Green I as intercalator, or TaqMan probe if available.
LibreBiotech setup. Create Process in production Study. Lot number format: RX-YYYYMMDD-NN (RTX / date / batch-of-day).
Expected contrast vs. procedures 59 (OpenTaq) and 65 (OpenVent): different inducer (IPTG like OpenVent, not aTC like OpenTaq/Bst-LF); different induction temperature and duration (overnight at 18°C vs. 3–4 h at 37°C); different selection antibiotic (kanamycin vs. ampicillin). The cold induction reduces aggregation of RTX into inclusion bodies — a common problem with engineered polymerases at 37°C.
- Day 0: plate transformation → 37°C overnight.
- Day 1: pick colony → starter culture overnight at 37°C, 225 rpm.
- Day 2 morning (~3 h): 1:200 dilution into 50 mL media, grow at 37°C to OD600 0.4–0.6 (~1.5 h), induce with 1 mM IPTG, transfer to 18°C shaking incubator for 16–18 h overnight induction (differs from OpenTaq/Bst-LF 3 h at 37°C).
- Day 3 morning (~1 h): harvest, wash, aliquot.
- Day 3 → Day 4/5: evaporative drying at 37°C static, 1–2 days.
- Day 5: seal, heat-inactivate, RT-qPCR activity QC, store.
- Total active time: ~5 hours across 5 days (the overnight cold induction adds an extra calendar day but minimal active time).
-
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-RT / post-PCR sets ideally -
Spectrophotometer
Measurement
Specs: OD600 -
Vortex mixer
Mixing
Specs: Standard benchtop -
Biosafety cabinet
Safety
Specs: Class II Type A2 or equivalent; PC1
All open-culture steps -
Desiccator jar
Storage
Specs: ~118 mL sealed plastic or glass jar
Reusable -
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
Initial growth phase (~1.5 h) -
Shaking incubator
Thermal regulation
Specs: 18°C, 225 rpm (must have active cooling)
Overnight induction — critical for RTX folding. Same unit as 37°C one if it can switch temperatures
-
E. coli BL21(DE3)
(NEB)
C2527IBacterial strain
Qty: 1 transformant
NEB C2527I or equivalent -
pET-28a
(Novagen) Expression plasmid
Qty: 1 ng
pET-28a vector with RTX insert from Ellington lab (Ellefson et al. 2016) or OBL derivative. Obtain via Addgene or direct from the lab -
Kanamycin
Antibiotic
Qty: 30 µg/mL final
pET-28a carries kanamycin resistance — differs from pATetO-based procedures which use ampicillin -
IPTG
Inducer
Qty: 1 mM final
1 M stock in water, filter-sterilised, single-use −20°C 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
Wash + 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 at induction. 1 mM standard for T7-promoter systems. |
iptg_induction_time_h |
number | — |
17
|
hour (UO:0000032) | Duration of IPTG-mediated expression at 18°C. 16–18 h standard; timing is flexible because low-temperature induction is slow and self-regulating. |
induction_temp_c |
number | — |
18
|
degree Celsius (UO:0000027) | Overnight induction temperature. CRITICAL — 18°C is what prevents RTX from aggregating into inclusion bodies. Higher temperatures (e.g. 37°C) dramatically reduce functional yield. |
pre_induction_temp_c |
number | — |
37
|
degree Celsius (UO:0000027) | Growth temperature before induction. Standard 37°C; culture transferred to 18°C at IPTG addition. |
harvest_od600 |
number | — |
0.5
|
— | OD600 at time of induction (transfer to 18°C). 0.4–0.6 window; midpoint 0.5. |
final_suspension_od600 |
number | — |
6.5
|
— | OD600 of concentrated suspension before aliquotting. |
rt_qc_temp_c |
number | — |
50
|
degree Celsius (UO:0000027) | Reverse transcription temperature in the activity QC assay. 50°C optimal for RTX. |
rt_qc_time_min |
number | — |
10
|
minute (UO:0000031) | Reverse transcription duration in QC. 10 min sufficient for short targets. |
heat_inactivation_temp_c |
number | — |
60
|
degree Celsius (UO:0000027) | Heat inactivation of GMO host. 60°C × 10 min; RTX retains function at this temperature. |
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 0.1.0)
Transform BL21(DE3) competent cells with 1 ng of pET-28a-RTX plasmid, using kanamycin 30 µg/mL as the selection antibiotic. Follow LibreBiotech procedure 60. Plate onto LB + kanamycin agar prepared per procedure 63.
Incubate transformation plates inverted at 37°C for 14–16 hours.
Pick one well-isolated colony into 3–5 mL Superior Broth + 30 µg/mL kanamycin. Grow overnight at 37°C, 225 rpm.
Prepare a glycerol stock per LibreBiotech procedure 61. All future RTX batches start from this stock.
Dilute the overnight culture 1:200 into 50 mL Superior Broth + 30 µg/mL kanamycin in a 250 mL flask.
Incubate at 37°C, 225 rpm, until OD600 reaches 0.4–0.6 (~1.5 h). Measure per LibreBiotech procedure 62. Do not exceed 0.7 — over-grown cultures at time of cold shift yield more inclusion bodies.
Induce expression by adding IPTG to 1 mM final concentration.
Immediately transfer the flask to a shaking incubator pre-equilibrated at 18°C, 225 rpm. Continue shaking overnight (16–18 h). The cold temperature slows translation, reducing aggregation of RTX into inclusion bodies — critical for functional yield.
The next morning, harvest cells by centrifugation at 9,000 × g for 1 minute at room temperature. Do NOT warm the culture before harvest — transition directly from 18°C to centrifugation.
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.
Perform RT-qPCR activity QC on one randomly-chosen tube per batch. Rehydrate in an RT-qPCR master mix with validated control RNA template and matched primers. Run: 50°C × 10 min (RT) → 95°C × 3 min → 45 cycles of 95°C × 15 s / 55°C × 30 s / 60°C × 60 s. Expected: clear amplification curve in positive control; no amplification in NTC. Discard 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 RX-YYYYMMDD-NN), Sample records per strip, Assay record with RT-qPCR amplification curve + melt curve attached.
Expected outcome (per 50 mL induced culture). ~8 aliquot strips (64 single-use RT-qPCR reactions' worth). Functional in one-enzyme RT-qPCR; detection limit around 5,000 copies per reaction (per the reference paper, using SARS-CoV-2 N1 assay with EvaGreen).
Storage. Sealed desiccator jar with fresh Drierite, dark cabinet, 20–25°C. Refrigerated storage at 4°C extends shelf life.
Activity QC (per batch, mandatory). Rehydrate one tube in a RT-qPCR master mix with a validated control RNA template and matched primers. Run RT-qPCR with EvaGreen or SYBR Green I detection: typical cycling 50°C × 10 min (reverse transcription) → 95°C × 3 min → 45 cycles of 95°C × 15 s / 55°C × 30 s / 60°C × 60 s → melt curve analysis. Expected: clear amplification curve for positive control, Cq ~30–35 for 10⁵ copies input; NTC should show no amplification or very late Cq (>40). Discard batch if QC fails.
One-enzyme vs. two-enzyme RT-PCR. The key value of RTX is that you do NOT need a separate reverse transcriptase. This simplifies reaction setup, reduces reagent cost, and eliminates the thermal-cycling gap between RT and PCR. For comparison:
- Standard two-enzyme (MMLV + Taq): RT at 37°C × 30 min → 95°C × 3 min inactivation → PCR cycles. ~60 min total.
- RTX one-enzyme: RT at 50°C × 10 min → PCR cycles. ~45 min total. Single master mix.
Limitations. RTX is less efficient than dedicated reverse transcriptases like SuperScript IV for long reverse transcripts. Not recommended for full-length mRNA / cDNA library preparation; suitable for targeted RT-qPCR of short amplicons (<500 bp).
Troubleshooting.
| Symptom | Likely cause | Fix |
|---|---|---|
| Induced cells grow but RT activity absent | 18°C induction too cold for your specific incubator | Verify chamber temperature; some incubators fail below ambient. Try 22–25°C overnight as fallback |
| Inclusion bodies visible in cell pellet (dense white band post-spin) | 37°C overnight induction attempted | Strictly 18°C for overnight; reduces aggregation |
| RT-qPCR fails on positive control | Template RNA degraded | Include RNase inhibitor in QC master mix; fresh aliquot of control RNA |
| Late Cq on positive control (>35 at 10⁵ copies) | Low RTX activity in batch | Retest fresh induction; verify IPTG stock; confirm 16 h minimum induction |
| NTC shows amplification | Cross-contamination (common with RT-PCR) | Dedicated pre-RT / post-PCR workflow areas; fresh pipettes; UV-decontaminate benches |
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
- Ellefson JW, Gollihar J, Shroff R, Shivram H, Iyer VR, Ellington AD (2016). Synthetic evolutionary origin of a proofreading reverse transcriptase. Science 352(6293):1590–3. (Original RTX engineering paper) DOI paper
- LibreBiotech procedures 60–64 (transformation, glycerol stock, OD600, LB plates, desiccator drying). Link protocol
- LibreBiotech procedure 59 — OpenTaq Cellular Reagent Production (workflow reference; note induction differences). Link protocol
- Addgene — pET-28a vector backbone (Novagen), required for RTX-insert constructs. Link paper