Table 1 Names and descriptions of each no gRNA control and kill switch strain. Cas9-expressing plasmids include a low (~5)-copy pSC101 origin of replication and gRNA-expressing plasmids include a medium (~20)-copy p15A origin of replication.
From: Genetically stable CRISPR-based kill switches for engineered microbes
| Â | Genetic parts | Knockouts | Figures | ||||
|---|---|---|---|---|---|---|---|
Strain name | gRNAs | cas9 | tetR | infA | tlpA | ||
No gRNA controls | |||||||
Control | None | Plasmid | Unoptimized | None | None | None | Fig. 1c |
Control | None | 4 Integrations | Unoptimized | None | None | None | Fig. 1h |
Control | None | 4 Integrations | Unoptimized | Optimized | None | None | Fig. 3a |
Control | None | 4 Integrations | Unoptimized | Optimized | None | ΔrecA ΔpolB ΔdinB ΔumuBC | |
aTc-inducible kill switches | |||||||
groL-1 | groL-1 | Plasmid | Unoptimized | None | None | None | Fig. 1c |
groL-2 | groL-2 | Plasmid | Unoptimized | None | None | None | Fig. 1c |
ile-1 | ile-1 | Plasmid | Unoptimized | None | None | None | Fig. 1c |
ile-2 | ile-2 | Plasmid | Unoptimized | None | None | None | Fig. 1c |
rrs-1 | rrs-1 | Plasmid | Unoptimized | None | None | None | Fig. 1c |
rrs-2 | rrs-2 | Plasmid | Unoptimized | None | None | None | Fig. 1c |
Int X1 | ile-2 | 1 Integration | Unoptimized | None | None | None | Fig. 1f |
Int X2 | ile-2 | 2 Integrations | Unoptimized | None | None | None | Fig. 1f |
Int X3 | ile-2 | 3 Integrations | Unoptimized | None | None | None | Fig. 1f |
Int X4 | ile-2 | 4 Integrations | Unoptimized | None | None | None | |
Initial 2-gRNA | groL-2 and ile-2 | 4 Integrations | Unoptimized | None | None | None | Fig. 1h |
Optimized 2-gRNA | Optimized groL-2 and ile-2 | 4 Integrations | Unoptimized | None | None | None | |
ABX-independent 2-gRNA | Optimized groL-2 and ile-2 | 4 Integrations | Unoptimized | Unoptimized | None | None | Fig. 2b |
CRISPRks | Optimized groL-2, ile-2, and rrs-2 | 4 Integrations | Unoptimized | Optimized | None | None | |
CRISPRks Δrpdu | Optimized groL-2, ile-2, and rrs-2 | 4 Integrations | Unoptimized | Optimized | None | ΔrecA ΔpolB ΔdinB ΔumuBC | |
2-input kill switches | |||||||
Initial 2-input CRISPRks Δrpdu | Optimized groL-2, ile-2, and rrs-2 | 4 Integrations | Unoptimized | Optimized | Present | ΔrecA ΔpolB ΔdinB ΔumuBC | Fig. 4b |
[Optimized] 2-input CRISPRks Δrpdu | Optimized groL-2, ile-2, and rrs-2 | 4 Integrations | Optimized | Optimized | Present | ΔrecA ΔpolB ΔdinB ΔumuBC | |
