Table 1 Plasmids and strains used in this study
Plasmids/Strains | Characteristics | Source or reference |
|---|---|---|
Plasmids | Â | Â |
pSL1180 | ColE ori; Ampr | Pharmacia |
p15A-M6.8 | p15A ori; lacZ; miniHimar1 transposon elements; kmr | [26] |
pCM80 | Universal plasmid used in M. extorquens, with a PmxaF promoter; Tet r | [7] |
pCM157 | Contains the constitutively expressed cre gene; Tetr | [9] |
pLC291 | Contains PR/tetO inducible promoter; kmr | Â |
pCM80-mcr | mcr gene was connected next to the PmxaF promotor on pCM80; Tet r | This study |
pCM80-eGFP | egfp gene was connected next to the PmxaF promotor on pCM80; Tet r | This study |
pCM80-mcr-eGFP | egfp co-transcribed with mcr was linked to pCM80; Tet r | This study |
pLC-Cre | cre gene was connected next to PR/tetO inducible promoter on pLC292; kmr | This study |
pSL-TP | miniHimar1 transposon elements was linked to pSL1180; Ampr | This study |
pSL-TP-cre-km | cre and lox71/ lox66 sites were linked to pSL-TP; Ampr; kmr | This study |
pTP-cre-mcr-egfp | Contains p15A ori; miniHimar1 transposon elements; PR/tetO inducible promoter; cre- lox71/lox66 cassette; mcr-egfp; kmr | This study |
pTP-mcr-egfp | Contains p15A ori; miniHimar1 transposon elements; mcr-egfp; kmr | This study |
Strains | Â | Â |
M. extorquens AM1 | Original host for the production of 3-HP | [7] |
M. extorquens AM1(lacZ) | Transformant of M. extorquens AM1 with chromosomal insertion of lacZ gene by p15A-M6.8 | [26] |
M. extorquens AM1-TC | Plasmid pSL-TP-cre-km was transformed into M. extorquens AM1, kmr | This study |
M. extorquens AM1-T | M. extorquens AM1-TC was induced to perform recombination at the lox71 and lox66 sites, resulting in the removal of cre-km cassette | This study |
AM1-MG80 | Transformant of M. extorquens AM1 with plasmid-dependent mcr-egfp cassette introduced by pCM80-mcr-egfp | This study |
AM1-M80 | Transformant of M. extorquens AM1 with plasmid-dependent mcr introduced by pCM80-mcr | This study |
AM1-MGTC | First-round mcr chromosomal integration recombinant generated by transforming pTP-cre-mcr-egfp into M. extorquens AM1, containing the mcr-egfp cassette and inducible cre. | This study |
AM1-MGT | Single mcr chromosomal integration recombinant generated by transforming pTP-mcr-egfp into M. extorquens AM1, containing the mcr-egfp cassette without inducible cre. | This study |
AM1-2MGTC | Second-round mcr chromosomal insertion recombinant obtained by re-transformation of pTP-cre-mcr-egfp into the AM1-MGTC after induction. | This study |
AM1-DMG | Combination expression recombinant obtained by transforming pTP-cre-mcr-egfp into AM1-MG80 | This study |
AM1-2DMG | Combination expression recombinant obtained by transforming pCM80-mcr-egfp into AM1-2MGTC | This study |