Protocols#

Where a published protocol was modified, the modification and its reason are given.

Strains and media#

E. coli DH10β for all cloning, BL21 (DE3) for expression of the shell variants, and the MutaT7 host strain for propagating the mutation plasmid and for the planned campaign. Cultures in 2×YT at 37 °C and 200 rpm unless stated. Antibiotics at 100 µg/mL ampicillin, 50 µg/mL kanamycin (200 µg/mL for high-stringency plates) and 25 µg/mL chloramphenicol. Glucose to 1 % (w/v) in expression cultures suppresses leaky T7 expression before induction. LB with 1 % agar plates carry the matching antibiotic for cloning and selection.

Transformation and recovery#

Chemically competent cells prepared by the calcium chloride method: overnight culture diluted into 50 mL, grown to OD₆₀₀ 0.5–0.6, chilled on ice 30 min, pelleted at 4000 × g, washed twice in ice-cold 100 mM CaCl₂ with a 30 min incubation between washes, resuspended in 5 mL CaCl₂ with 80 % glycerol, aliquoted and flash-frozen.

Practical notes

Cells are kept cold at all times, including during transport to the centrifuge.
About 20–30 µL of cells are used per transformation. One aliquot covers about five transformations; larger volumes do not improve efficiency.
The MutaT7 strain transforms poorly, so aliquots of 200 µL are prepared instead of 100 µL.
For low-efficiency assemblies, the recovery culture is pelleted and resuspended in ~50 µL before plating, instead of plating 400 µL.

Transformation: thaw 10 min on ice, add 2–5 µL DNA, 30 min on ice, heat shock 42 °C for 30 s, 5 min on ice, recover in 400 µL SOC at 37 °C for 1 h, plate.

Cloning#

Gibson assembly. 10–20 µL reactions, 0.05 pmol per fragment, insert:backbone molar ratios 1:1 to 3:1, 50 °C for 15–60 min.

Golden Gate. PaqCI or BsaI-HFv2 with NEBridge Ligase Master Mix, 15–30 µL, as a one-hour isothermal incubation at 37 °C or a cycled digestion–ligation programme. For the selection plasmids, whole-plasmid sequencing of the reaction mixture showed which junctions formed (Table S2).

PCR and verification. Inserts and backbones by PCR with Q5 or Phusion, 20–30 s per kb extension, purified by agarose gel (0.5 % in TAE, 130 V, 25 min) and column extraction. Constructs verified by Sanger sequencing of assembly junctions and by whole-plasmid sequencing (Microsynth; NightSeq for direct colony submission), alignments curated in Benchling.

Linearisation. pSC101 with EcoRI and BamHI; the MutaT7 backbone with PacI and SacI. Digests were run for 2–3 hours; these enzymes have little star activity, so the longer incubation reduces incomplete cutting without a cost.

Reading a linearisation gel

Supercoiled circular plasmid migrates faster than the same plasmid linearised. A strong upper band and a faint lower band therefore do not necessarily mean that the lower band is the cut product. The wrong band was excised once for this reason.

PCR-linearised backbones are DpnI-digested for 1 h at 37 °C to remove template.

Expression#

Primary culture 5 mL overnight, 37 °C, 200 rpm. Secondary culture 250 mL 2×YT with antibiotic and 1 % glucose, inoculated to OD₆₀₀ 0.05.

Induction conditions

Induce at OD₆₀₀ 0.4–0.6 with 0.1 mM IPTG, then move to 18 °C, 120 rpm, 18 h.

Earlier runs used 1 mM IPTG. Published protocols for this shell use 0.1 mM, and strong induction commonly leads to inclusion bodies. Glucose suppresses leaky expression before induction; IPTG takes effect once the glucose is consumed.

Aliquots are taken immediately before and after induction, normalised to OD, for later SDS-PAGE, so that protein loss can be located to a step.

Purification#

Lysis. Resuspend in 20 mM Tris-HCl pH 8.0, 150–500 mM NaCl. Chemical lysis with lysozyme and 1 mM PMSF, then sonication on ice (24 % amplitude, 2 s on / 4 s off, 5 min total on-time).
Clarify. 10 000 × g, 15 min, 4 °C. The pellet is kept, as most of the protein was found in it.
Pre-enrichment, one of two routes run on split aliquots:
PEG-8000/NaCl: to 10 % PEG and 0.5 M NaCl, 40 min on ice, 8000 × g 10 min.
Heat precipitation, 65 °C for 15 min in a water bath, exploiting the shell's thermostability; keep the supernatant.

Filter through 0.45 µm and 0.22 µm before further use. 4. Ni-NTA (where used). Gravity column, Ni-Sepharose 6 Fast Flow; 1 mL of a 50 % resin slurry per 4 mL of sample, 1 h at 4 °C, two washes of 1 mL at 20 mM imidazole pH 8, elution in 1 mL of 250 mM imidazole pH 8. For QtEnc-His this step discards the assembled species along with the insoluble fraction (Results), so pellet-derived material was taken forward without it. 5. Buffer exchange. Amicon 100 kDa cut-off, 4000 RCF, 4 °C. Pre-wash the filter three times with water and equilibrate in elution buffer first. 6. SEC, two tiers. A crude cut on HiPrep Sephacryl S-500 HR at 0.5 mL/min (1 mL fractions), then high-resolution SEC on Superose 6 10/300 GL at 0.3 mL/min, reduced from the standard rate to keep system pressure below 1.5 MPa. Buffer: 20 mM Tris, 200 mM NaCl, pH 8.0 with 0.02 % sodium azide, filtered through 0.45 µm and degassed 30 min. Fractions into 96-well plates and pooled by peak.

SEC notes

The fraction collector follows a snake pattern across the plate, so wells are counted up or down depending on the row letter.

Sodium azide is added to the SEC buffer, as buffer without it became contaminated with fungi.

Analysis#

SDS-PAGE. Precast gels, 165 V, 25–40 min. 10–20 µg total protein per lane for crude samples. Sample mixed with 4× loading buffer to 1×, 95 °C for 5 min.

Native PAGE. Blue Native and clear-native precast gels, no detergent in the sample buffer, which preserves assembled shells. The gels are fragile and are handled with water on the tray.

Anti-His immunoblot. Semi-dry transfer; with the high molecular weight setting for native gels.

DLS. Prometheus Panta and cuvette formats, samples below 1 mg/mL in SEC buffer. Protein concentration from A₂₈₀ with construct-specific extinction coefficients.

Stop-codon reversion assay#

Tests MutaT7 mutagenesis with two reporters: p_m004 (kanamycin-resistance reporter) and p_m008 (chloramphenicol-resistance reporter), each with an early stop codon.

Pick random colonies from selective plates and resuspend in 60 µL 2×YT; three independent colonies per experiment serve as biological replicates. Inoculate 15 µL into 5 mL 2×YT with 50 µg/mL ampicillin and one additive: 1 mM IPTG (induced), repressing agent (glucose at 0.5 % for the kanamycin reporter, 1 % 2,4-diacetylphloroglucinol (DAPG) for the chloramphenicol reporter where the selection plasmid is present, 1 % glucose where it is not), or none (basal). Grow overnight, plate 50 µL on LB agar with 50 or 200 µg/mL kanamycin, or 25 µg/mL chloramphenicol, and count colonies after 48 h at 37 °C (Fig S13).

Continuous culture#

Planned in a turbidostat (Pioreactor). The passaging and stringency schedule is given under planned campaign.

Co-encapsulation assays#

Planned for enriched shell alleles. Because the selection is satisfied by capture of either the guide or the nuclease, these assays distinguish the two:

Native PAGE, dual-stained: nucleic acid and protein in the same lane.
RNase challenge: encapsulated RNA is protected and free RNA is degraded. Tetter and co-workers used this assay in their packaging selection. It tests protection and not only binding.
Native PAGE with a fluorescent cargo, and SEC co-elution of cargo with the shell peak, for protein loading.
The stop-codon reversion assay above tests MutaT7 function and does not measure sequestration.