| Description | Sources |
---|---|---|
Strains | ||
E. coli 10-β | High efficiency strain ideal for cloning | C3019, NEB |
E. coli S17-1 | Conjugative donor strain | ATCC 47,055 |
C. sporogenes WT | Wild type strain, Clostridium sporogenes NCIMB 10,696 | NCIMB culture collection (UK) |
C. butyricum WT | Wild type strain, Clostridium butyricum DSM10702 | DSMZ-German Collection |
C. butyricumΔpyrE | C. butyricum with deletion of the PyrE loci | [28] |
C. butyricumINpyrE::ESO | C. butyricum with integration of the miniPc.b_fU-NY-ESO-1 into the PyrE loci | This work |
C. butyricumINpyrE::CDP-ESO | C. butyricum with integration of the miniPc.b_fU-CDP-NY-ESO-1 into the PyrE loci | This work |
C. sporogenes-NT | Non-toxic C. sporogenes NCIMB 10,696, via deletion of the putative Streptolysin S operon | [18] |
C. sporogenes-NTΔpyrE | C. sporogenes-NT with deletion of the PyrE loci | This work |
C. sporogenes-NTINpyrE::ESO | C. sporogenes-NT with integration of the miniPc.sp_fU-NY-ESO-1 into the PyrE loci | This work |
C. sporogenes-NTINpyrE::CDP-ESO | C. sporogenes-NT with integration of the miniPc.sp_fU-CDP-NY-ESO-1 into the PyrE loci | This work |
C. sporogenes-NTINpyrE::S1 | C. sporogenes-NT with integration of the miniPc.b_fU-S1 into the PyrE loci | This work |
C. sporogenes-NTINpyrE::CDP-S1 | C. sporogenes-NT with integration of the miniPc.b_fU-CDP-S1 into the PyrE loci | This work |
Plasmids | ||
pGG2121 | Golden Gate assembling and E. coli - Clostridium shuttle vector, Cmr | [27] |
pPromoters-SpnprM3-NY-ESO-1 | pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized NY-ESO-1 gene | This work |
pPromoters-SpnprM3-CDP-NY-ESO-1 | pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized CDP sequence and NY-ESO-1 gene | This work |
pPromoters-SpnprM3-S1 | pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized Spike_S1 sequence | This work |
pPromoters-SpnprM3-CDP-S1 | pGG2121 ligated with promoters, the nprM3 signal peptide, and the codon-optimized CDP and Spike_S1 gene sequence | This work |
pPIPL12-FnCas12a | Entry vector with tetracycline-Inducible expression of FnCas12a in E. coli and clostridia, Ermr | [28] |
pPfetFn-Target_v2 | Entry vector with gRNA expression for FnCas12a in E. coli and clostridia, Cmr | [28] |
pPfetFn-IncbPyrE::ESO | pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.b_fU-NY-ESO-1 expression cassette into C. butyricum PyrE loci | This work |
pPfetFn-IncbPyrE::CDP-ESO | pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.b_fU-CDP-NY-ESO-1 expression cassette into C. butyricum PyrE loci | This work |
pPfetFn-DspPyrE | pPfetFn-Target_v2 ligated with the donor DNA template and target for deleting pyrE gene in C. sporogenes-NT | This work |
pPfetFn-InspPyrE::ESO | pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.sp_fU-NY-ESO-1 expression cassette into C. sporogenes-NT PyrE loci | This work |
pPfetFn-InspPyrE::CDP-ESO | pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.sp_fU-CDP-NY-ESO-1 expression cassette into C. sporogenes-NT PyrE loci | This work |
pPfetFn-InspPyrE::S1 | pPfetFn-Target_v2 ligated with the donor DNA template and target for integrating the miniPc.b_fU-S1 expression cassette into C. sporogenes-NT PyrE loci | This work |
pPfetFn-InspPyrE::CDP-S1 | pPfetFn-Target_v2 ligaed with the donor DNA template and target for integrating the miniPc.b_fU-CDP-S1 expression cassette into C. sporogenes-NT PyrE loci | This work |