Table 1.
Bacterial strains and plasmids used in this study.
| Strains or plasmids | Characteristics | Source |
|---|---|---|
| Strains | ||
| E. coli MG1655 | F- lambda- rph-1 | CGSC 6300 |
| H. bluephagenesis TD01 | H. bluephagenesis wild type | Cai et al., 2011 |
| E. coli MG1655AX01 | MG1655 ΔtorS::p103-hem1 | This study |
| E. coli MG1655AX02 | MG1655 ΔlacZ::T7 RNAP, ΔtorS::pT7-hem1 | This study |
| E. coli MG1655AX03 | MG1655 with the plasmid pLTT05 | This study |
| Plasmids | ||
| pcrRNA-P | crRNA-pyrF, pUC origin, kan | This study |
| p46Cas9 | Expressing λ-Red and SpCas9 | This study |
| p46Cpf1 | Expressing λ-Red and wild-type FnCas12a (FnCpf1) | This study |
| p46Cpf1-OP1 | Expressing λ-Red and codon-optimized FnCas12a (type 1) | This study |
| p46Cpf1-OP2 | Expressing λ-Red and another codon-optimized FnCas12a (type 2) | This study (Addgene #98592) |
| pTs-P | sgRNA-pyrF, ΔpyrF::gfp | This study |
| pTs-PL | sgRNA-lacZ, ΔlacZ::aadA sgRNA-pyrF, ΔpyrF::gfp | This study |
| pTs-GLP | sgRNA-galK,ΔgalK::rfp sgRNA-lacZ,ΔlacZ::aadA sgRNA-pyrF, ΔpyrF::gfp | This study |
| pTc-P | crRNA-pyrF, ΔpyrF::gfp | This study |
| pTc-P-50bp | crRNA-pyrF, ΔpyrF::gfp with 50 bp homology arms | This study |
| pTc-P-100bp | crRNA-pyrF, ΔpyrF::gfp with 100 bp homology arms | This study |
| pTc-G | crRNA-galK, ΔgalK::rfp | This study |
| pTc-G2 | crRNA-galK2, ΔgalK::rfp | This study |
| pTc-A | crRNA-araD, ΔaraD::rpsl | This study |
| pTc-A2 | crRNA-araD2, ΔaraD::rpsl | This study |
| pTc-M | crRNA- mutS, ΔmutS::tcr | This study |
| pTc-L | crRNA-lacZ, ΔlacZ::aadA | This study |
| pTc-GL | crRNA-galK, ΔgalK::rfp crRNA-lacZ, ΔlacZ::aadA | This study |
| pTc-GP | crRNA-galK, ΔgalK::rfp crRNA-pyrF, ΔpyrF::gfp | This study |
| pTc-PL | crRNA-lacZ, ΔlacZ::aadA crRNA-pyrF, ΔpyrF::gfp | This study |
| pTc-GLP | crRNA-galK,ΔgalK::rfp crRNA-lacZ,ΔlacZ::aadA crRNA-pyrF, ΔpyrF::gfp | This study |
| pTc-arrayLP | crRNA-lacZ-pyrF, ΔlacZ::aadAΔpyrF::gfp | This study |
| pTc-arrayPL | crRNA-pyrF-lacZ, ΔlacZ::aadAΔpyrF::gfp | This study |
| pTc-torS-p103-hem1 | crRNA-torS, ΔtorS::p103-hem1 | This study |
| pTc-lacZ-T7RNAP-torS-pT7-hem1 | crRNA-lacZ, ΔlacZ::T7 RNAP crRNA-torS, ΔtorS::pT7-hem1 | This study |
| pLTT05 | Expressing T7 RNA polymerase and ALA synthase (hem1) | Li et al., 2016 |
| pTD-Cas12a | Expressing codon-optimized FnCas12a (type 2) | This study |
| pTtd-prpC | crRNA-prpC, ΔprpC | This study |
| pTtd-prpC-PM | crRNA-prpC, prpC with point mutation | This study |
kan, kanamycin resistance gene; rfp, red fluorescent protein gene; aadA, spectinomycin resistance gene; gfp, green fluorescent protein gene; rpsl, streptomycin resistance gene; tcr, tetracycline resistance gene; hem1, mitochondrial 5-aminolevulinic acid synthase gene. sgRNA-pyrF, sgRNA targeting the pyrF locus; sgRNA-galK, sgRNA targeting the galK locus; sgRNA-lacZ, sgRNA targeting the lacZ locus; crRNA-pyrF, crRNA targeting the pyrF locus; crRNA-galK and crRNA-galK2, crRNA targeting the galK locus; crRNA-araD and crRNA-araD2, crRNAs targeting the araD locus; crRNA-mutS, crRNA targeting the mutS locus; crRNA-lacZ, crRNA targeting the lacZ locus; crRNA-torS, crRNA targeting the torS locus; crRNA-prpC, crRNA targeting the prpC locus; crRNA-lacZ-pyrF and crRNA-pyrF-lacZ, crRNAs in a CRISPR array targeting the galK locus and the lacZ locus. ΔpyrF::gfp, editing template with two flanking 500 bp sequences homologous to the pyrF locus with a gfp insertion; ΔgalK::rfp, editing template with two flanking 500 bp sequences homologous to the galK locus with an rfp insertion; ΔaraD::rpsl, editing template with two flanking 500 bp sequences homologous to the araD locus with an rpsl insertion. ΔlacZ::aadA, editing template with two flanking 500 bp sequences homologous to the lacZ locus with an aadA insertion; ΔmutS::tcr, editing template with two flanking 500 bp sequences homologous to the mutS locus with an tcr insertion; ΔlacZ::T7 RNAP, editing template with two flanking 500 bp sequences homologous to the lacZ locus with a T7 RNA polymerase gene insertion; ΔprpC, editing template with two flanking 500 bp sequences homologous to the prpC locus. p103-hem1, hem1 controlled by the 103 promoter; pT7-hem1, hem1 controlled by the T7 promoter.