2026

Notebook#

Condensed from the lab journal kept from 13 May 2026. From late July, shell expression and purification and construction of the selection circuit were carried out in parallel.

May#

Competent cells (DH10β and BL21 (DE3)) and buffer stocks were prepared and the expression backbone was transformed. An early plating was done on kanamycin although the plasmid carried ampicillin resistance, and no colonies grew. Gibson assemblies of the first shell constructs were performed, and colonies were picked and sent for sequencing.

June#

First large cultures, induced with 1 mM IPTG at 18 °C overnight.

The available Cytiva Ni-NTA protocol was written for pre-packed HiTrap columns, so a protocol for columns packed in-house was assembled from it, a published methods section and local practice. First SDS-PAGE gels showed protein of approximately the expected mass.

July#

Size exclusion of the first purified material gave a shallow peak around 8 mL and substantial material at 16, 18 and 20 mL. An assembled cage is expected to elute near 8 mL. DLS of the pooled fractions did not show particles of the expected size.

Fractions had been pooled from 16 mL onward, on the assumption that the early peak was negligible. SEC and DLS both indicated a problem with cage assembly. Hypotheses were listed and tested one at a time.

Changes: induction was reduced to 0.1 mM IPTG; a 100 kDa Amicon filter was introduced to remove monomer and free reporter; aliquots were taken at every purification step for gel analysis; clear-native PAGE was added to detect the assembled species directly; anti-His immunoblotting was added to test whether the tag was accessible.

Addgene bacterial stabs of the dCas9 and MutaT7 source plasmids arrived at the end of the month and were streaked out.

August#

Purification. PEG precipitation and heat precipitation were run in parallel on split aliquots. A280 measurements and gels at each step located most of the protein in the pellet after lysis. SEC of heat-precipitated material showed no peak at 8–10 mL. DLS of pooled fractions indicated a polydisperse, dilute sample; some individual measurements suggested particles of the expected size, but the pooled measurements did not.

Circuit construction. dCas9 was amplified from the Addgene plasmid. pSC101 was linearised by restriction digest and by PCR. Assembly of the selection constructs (the guide truncation series and the non-targeting control) started with Gibson assembly and continued with Golden Gate assembly using synthetic fragments.

  • Early Gibson reactions gave no colonies, while a re-transformation control grew, which located the problem in the assembly.
  • The first s002_20nt clones sequenced as backbone plus an unrelated gBlock.
  • m004 and m005 first sequenced as empty backbone and were later recovered and verified.
  • MutaT7 competent cells transformed poorly, and the aliquot size was increased.

By the end of the month the shell expression constructs f008–f011 were sequence-confirmed. The s002 guide series remained in assembly (Table S2).

September#

Remaining fragments were amplified by PCR and gel-extracted. Repeated gel smearing was traced to reaction volume and run time. Kanamycin plates were prepared at 50 and 200 µg/mL, and with nitrotetrazolium blue for a colorimetric viability readout, for the selection screen.

The s002 sequences are confirmed. Assembly of the mutation plasmid series continues.

Nine constructs have been verified and a working expression protocol was established. Cage assembly has not been confirmed and the selection has not been run (Results).

The stop-codon reversion assay confirmed MutaT7 activity, and the purification data localised assembled QtEnc-His to the insoluble fraction. Both are described in Results; raw traces and gels are in the supplementary figures.