TABLE 4.
Strains, plasmids, and primers used in this study
| Strain, plasmid, or primer | Description | Purpose | Source |
|---|---|---|---|
| Strains | |||
| Methanosarcina acetivorans C2A | |||
| 394 | Δhpt::φC31 int, att:pJA2 | Isoprene production (att:ispS) | This study |
| 452 | Δhpt::φC31 int, att:pNB730 | Vector-only control (att:pNB730) |
(55) |
| Methanosarcina barkeri | |||
| 396 | Δhpt::φC31 int, att:pJA2 | Isoprene production | This study |
| 459 | Δhpt::φC31 int, att:pNB730 | Vector-only control (att:pNB730) |
(31) |
| Escherichia coli | |||
| 3 | F′proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10 (TetR)/fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 endA1 thi-1 hsdR17 | Cloning and plasmid propagation | New England Biolabs |
| 134 | F′ proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10 (TetR)/fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 endA1 thi-1 hsdR17/ pNB730 | Production of pNB730 plasmid | (31) |
| 453 | F′ proA+B+ lacIq Δ(lacZ)M15 zzf::Tn10 (TetR)/fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80Δ(lacZ)M15 gyrA96 recA1 endA1 thi-1 hsdR17/ pJA2 | Expression of ispS in M. acetivorans | (55) |
| Plasmids | |||
| pNB730 | pUC ori bla PmcrB pac(opt) φC31 attB | Methanosarcina spp. integration and expression vector | (31) |
| pJA2 | pNB730 ispSΔ1−13 | Integration of ispS into genome for constitutive expression of isoprene synthase | (55) |
| Primers and DNA strings | |||
| sNB19 | GenBank accession no. MW295460 (see the supplemental material) | Synthetic optimized Populus alba ispS (isoprene synthase) | This study |
| oNB568 | ATTAAGGAGGAAATTCATATGTCCGTTTCCACCGAAAATGT | ispSΔ1−13 DNA string amplification and cloning, fwd | (55) |
| oNB576 | CGAGGGCCCAAGCTTGGATCCTCATCTTTCAAAAGGAAGAATAG | DNA string amplification and cloning, rev | (55) |
| oNB729 | CATATGCCTGACGACCTCATTA | RNA polymerase, rpoA1 qRT fwd housekeeping gene | This study |
| oNB730 | GAATTTGATTTCGAGCTGTTCC | RNA polymerase, rpoA1 qRT rev housekeeping gene | This study |
| oNB733 | GTTTACAAAAGTAGCTGCAAGGGTA | Methyl-coenzyme M reductase, mcrB qRT fwd | This study |
| oNB734 | ATACAAATTCTACAAGGCAAACGAC | Methyl-coenzyme M reductase, mcrB qRT rev | This study |
| oNB735 | GGATTCGATGCAGTTACCAAA | ispS qPCR primer, fwd | This study |
| oNB736 | TGCTTCCTGGCTAACTTCAAA | ispS qPCR primer, rev | This study |
| oNB930 | CCGTGCCTGATGTCGACGAA | HMG-CoA synthase fwd | This study |
| oNB931 | TGGAGGGATCTACGCCGCTT | HMG-CoA synthase rev | This study |
| oNB932 | GCCGGCCTTCTGAAAGTAAACG | HMGR Fwd | This study |
| oNB933 | TCCGCGGTTTACACTGGCAA | HMGR rev | This study |
| oNB934 | CCCGTGTGCGGGTGGAATTA | Mevalonate kinase fwd | This study |
| oNB935 | ACCACTGCGGAGATATAAGGATGT | Mevalonate kinase rev | This study |
| oNB936 | GAGGCAGCGCCATTACCGAT | Isopentenyl phosphate kinase fwd | This study |
| oNB937 | GCCTGAAACTTCCCGCGCAA | Isopentenyl phosphate kinase rev | This study |
| oNB938 | CAGCCAGAGAGCCGCAATCG | Isopentenyl-diphosphate δ-isomerase fwd | This study |
| oNB939 | CCGTAGACAAAGGCGTTCGGA | Isopentenyl-diphosphate δ-isomerase rev | This study |
| oNB940 | GCTCATGCACGAGGTGCTCT | Pyruvate synthase fwd | This study |
| oNB941 | GCACTGACTGCCCTGTTTGC | Pyruvate synthase rev | This study |
| oNB942 | TCATGCGTGCCTGCAGAGAG | Pyruvate carboxylase fwd | This study |
| oNB943 | GCCTCATCGGCATACTTGGCA | Pyruvate carboxylase rev | This study |
| oNB944 | CCGAACTGGAACCTGGCGAA | Malate dehydrogenase fwd | This study |
| oNB945 | TGCCTGCATGAGGTCAAGGG | Malate dehydrogenase rev | This study |
| oNB946 | TCCTCGACCTGCCTATCGGT | Fumarate hydratase fwd | This study |
| oNB947 | GGTCGGCTGGAACCTCAACC | Fumarate hydratase rev | This study |