Plasmids#
Two plasmids, co-resident in a MutaT7 host strain. The division between them is a containment decision, not a convenience.
The mutagenesis plasmid#
Carries two things: the MutaT7 fusion — a T7 RNA polymerase joined to a nucleotide deaminase — and the QtEncapsulin open reading frame flanked by a T7 promoter and a T7 terminator.
Because the fusion tracks processively along whatever sits behind a T7 promoter and stops at the terminator, that cassette is the only hypermutated sequence in the cell. The host genome, replicated by its own high-fidelity machinery, is unaffected.
Fig 1. The deaminase fusion tracking along the T7-flanked encapsulin cassette, and stopping at the terminator.
Built on the pDB004 / pDB006 backbones by Golden Gate assembly. Source plasmids for the MutaT7 machinery came from Addgene as bacterial stabs.
The selection plasmid#
Carries dCas9, the sgRNA cassette and the kanamycin-resistance gene containing the targeted site. Built on a pSC101 backbone by Gibson assembly, with dCas9 amplified out of an Addgene source plasmid.
The guide is supplied as a truncation series so the selection set-point can be chosen empirically:
| Construct | Complementarity | Purpose |
|---|---|---|
s002_NT |
none | Non-targeting control — defines unrepressed output |
s002_10nt |
10 nt | Weakest repression |
s002_11nt |
11 nt | |
s002_14nt |
14 nt | |
s002_17nt |
17 nt | |
s002_20nt |
20 nt | Full complementarity, strongest repression |
All six are sequence-confirmed. See Mechanism for why complementarity, rather than an inducer, is the knob.
Compatibility and copy number#
The two plasmids carry different origins and different resistance markers so they can be maintained together under dual selection. The kanamycin marker on the selection plasmid is not a maintenance marker — it is the selection, which means the plasmid's own retention and the phenotype being selected are the same signal. Maintenance of the mutagenesis plasmid is enforced separately.
The escape argument, in one line
dCas9 and the sgRNA sit on the non-mutable plasmid because any mutation that reduced their expression would restore resistance without improving encapsulation — and would be enriched just as fast as a genuine improvement.
This does not make them immune to host-polymerase error, only to hypermutation. Both plasmids are re-sequenced periodically during passaging.
Provenance#
Source plasmids obtained from Addgene: pdCas9-bacteria (dCas9), and
pSC101-T7-T3RNAP and the pDB series (MutaT7 machinery). Expression constructs
are built on pET-Duet-1. Full maps and sequences are maintained in Benchling;
see Attributions for what came from where.