Protocols#

Where a published protocol was modified, the modification and its reason are given. Several of these were painful to arrive at.

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.

Things that cost us time

Everything stays cold. Including on the way to the centrifuge.
Use ~20–30 µL of cells per transformation, not a whole aliquot. One aliquot covers about five transformations; using more does not help.
The MutaT7 strain transforms poorly. Make 200 µL aliquots for it, not 100 µL.
For low-efficiency assemblies, spin the recovery culture down and resuspend in ~50 µL before plating, rather than 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. A one-hour isothermal incubation at 37 °C worked better for us than short cycled programmes.

Linearisation. pSC101 with EcoRI and BamHI; the MutaT7 backbone with PacI and SacI. Run digests 2–3 hours — these enzymes have little star activity, so the extra time is free insurance against incomplete cutting.

Reading a linearisation gel

Supercoiled circular plasmid migrates faster than the same plasmid linearised. A strong high band and a faint low band does not automatically mean the low band is your cut product. We cut the wrong band at least once on this reasoning.

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 — the current 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 over-strong induction is a standard route to inclusion bodies. The glucose suppresses leaky expression before induction; note that IPTG only takes effect once the glucose is consumed.

Aliquots are taken immediately before and after induction, normalised to OD, for later SDS-PAGE — this is what lets a loss be located later instead of guessed at.

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.
Clarify. 10 000 × g, 15 min, 4 °C. Keep the pellet — most of the protein turned out to be 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, exploiting the shell's thermostability; keep the supernatant.
Ni-NTA. Gravity column, 1.5 mL Ni-NTA Sepharose, 20 mM imidazole wash, 250 mM imidazole elution.
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.
SEC. Superose 6 10/300 GL at 0.3 mL/min — reduced from the standard rate to keep system pressure below 1.5 MPa — in 20 mM Tris, 200 mM NaCl, pH 8.0 with 0.02 % sodium azide, filtered and degassed 30 min. 0.35 mL fractions into 96-well plates.

Two SEC gotchas

The fraction collector follows a snake pattern across the plate — count up or down depending on the row letter, or you will pool the wrong wells.

Add sodium azide to the SEC buffer. We lost buffer to fungal contamination without it.

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.

Clear-native PAGE. No detergent in the sample buffer — this is what resolves assembled shells. The gels are fragile; handle with water on the tray.

Anti-His immunoblot. Semi-dry transfer; use the high molecular weight setting when transferring from native gels.

DLS. Prometheus Panta and cuvette formats. Sample below 1 mg/mL.

Continuous culture#

Turbidostat (Pioreactor). See Evolution cycles for the passaging and stringency schedule. Not yet run.

Co-encapsulation assays#

Designed, being finalised. Because the selection can be satisfied by capturing either the guide or the nuclease, these assays exist to tell the two apart:

Native PAGE, dual-stained — nucleic acid and protein in the same lane.
RNase challenge — encapsulated RNA is protected, free RNA is degraded. This is the assay Tetter and co-workers escalated to drive their packaging selection, and the one that tests protection rather than mere binding.
SEC co-elution with a fluorescent cargo.
Stop-codon reversal assay for functional sequestration.