2026 Laboratory Protocol

Use ISCro4(S30T/P54Q/S243H) as the parent, pID05 as the expression and mutagenesis vector, and pUC19-d-T as the substrate plasmid. Compare the parent, 10 candidate variants, and the E60Q negative control.

Purpose and materials：Prepare pUC19-d-T, pID05, and sequence-verified pID05 variants from separate E. coli DH5α cultures for cloning and sequencing. Use a silica-column plasmid miniprep kit, microcentrifuge, sterile tubes, and a DNA quantification instrument. The example below follows the QIAprep Spin Miniprep high-yield workflow; use its buffer volumes only with the corresponding kit.

1. Grow a single colony in 5 mL 2×YT with the plasmid-specific antibiotic for 12–16 h at 37 °C. Pellet the culture at approximately 6,800 × g for 3 min and discard the supernatant.

2. Resuspend completely in 250 μL Buffer P1 containing RNase A. Add 250 μL Buffer P2, mix gently by inversion, and lyse for no longer than 5 min. Avoid vortexing after lysis.

3. Add 350 μL Buffer N3 and immediately invert to mix. Centrifuge for 10 min at approximately 17,900 × g. Transfer the clear supernatant to the spin column without disturbing the pellet.

4. Centrifuge for 30–60 s and discard the flow-through. Wash with 500 μL Buffer PB, followed by 750 μL ethanol-supplemented Buffer PE, centrifuging after each wash. Perform an additional 1-min dry spin.

5. Transfer the column to a clean tube. Add 60 μL Buffer EB to the membrane, wait 1 min, and centrifuge for 1 min to elute DNA.

6. Measure DNA concentration and record sample identity. Use purified DNA for Sanger sequencing of assembly junctions and the ISCro4 coding sequence, including the parent substitutions and each intended additional mutation. Store aliquots at −20 °C.

Purpose and materials： Assemble the DNA fragments needed for pUC19-d-T, pID05, or the revised expression and reporter constructs. Required materials include a linear vector, purified insert fragments, a 2× Gibson Assembly master mix, nuclease-free water, and competent E. coli DH5α. The reaction below is an example for NEB Gibson Assembly Master Mix.

1. Design adjacent fragments with approximately 20–40 bp terminal overlaps. Generate fragments with a high-fidelity polymerase and linearize the vector by PCR or restriction digestion. Check fragment sizes by agarose gel electrophoresis, purify, and quantify. Where PCR uses a methylated parental plasmid, treat with DpnI before purification to reduce template carryover.

2. Set up a 20 μL reaction with 10 μL 2× master mix, DNA fragments, and water to volume. For two or three total fragments, use 50–100 ng vector and a two- to threefold molar excess of each insert; keep total DNA at 0.02–0.5 pmol. Calculate insert mass as vector mass × (insert length/vector length) × desired molar ratio. For four to six total fragments, use approximately equimolar fragments with total DNA at 0.2–1.0 pmol.

3. Incubate at 50 °C for 15 min for two or three fragments, or 60 min for four to six fragments. Count the vector as one fragment.

4. Transform 2 μL reaction into competent DH5α using the supplier’s transformation and recovery instructions. Plate on LB agar with the antibiotic matching the vector’s selectable marker. Include a vector-only assembly control.

5. Screen colonies by PCR across assembly junctions. Purify candidate plasmids using Section 4.6 and confirm junctions, regulatory elements, and the relevant coding sequence by Sanger sequencing. For pID05 mutants, verify the intended substitution while retaining the parent S30T/P54Q/S243H background. Confirm the donor and target sites and the intervening deletion substrate in pUC19-d-T. Use sequence-verified constructs for subsequent assays.

Construct the designed plasmids using Gibson assembly and then perform transformation and clone propagation. Confirm the correctness of the construct by Sanger sequencing before starting the rest of the experiments.

Remove the ISCro4 expression cassette from the single-plasmid system. Observe the growth of cells carrying this control under different chloramphenicol conditions to determine whether the resistance readout has background. Evaluate the first Design of the assay.

Biomni can help with the selection of candidates based on deep mutational scanning data and primer design. Introduce each candidate substitution independently into the parental pID05 background, construct the E60Q control, and verify the resulting sequences.

Cotransform each expression plasmid with pUC19-d-T into E. coli. Select three independent colonies for each variant, culture them, normalise according to OD600, and prepare samples for qPCR.

Use qPCR-A-F/-R and qPCR-AmpR-F/-R to detect the signal of the area targeted for deletion and the plasmid backbone, respectively. Carry out 2 technical replicates per sample and record Cq values.

Calculate ΔCq = Cq(qPCR-A) - Cq(qPCR-AmpR). Summarise the results of candidates and parents using independent colonies as the unit of comparison. Based on the report, variants with a higher ΔCq than the parent are positive candidates; this index is relatively lower for plasmid DNA deletion. Save sequencing results, raw qPCR data and analysis files.