Table 2 Strains and plasmids used in this study
From: Engineering Bacillus megaterium for production of functional intracellular materials
Strain/plasmid name | Genotype/description | Source/references |
|---|---|---|
Strain | ||
 E. coli XL1BLue | recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F′ proAB lacI q lacZ ΔM15 Tn10 (Tet R )] | Stratagene |
 B. megaterium PHA05 | PHA-negative mutant of 11561 (ATCC); Δ(phaP-phaC); Emr | [41] |
 B. megaterium PHA05 ΔspoIIE | Sporulation-negative mutant of PHA05; Δ(phaP-phaC) ΔspoIIE; EmR CmR | This study |
Plasmid | ||
 pUC57-phaA | Codon optimized phaA in pUC57, AmpR | GenScript |
 pUC57-phaB | Codon optimized phaB in pUC57, AmpR | GenScript |
 pUC57-phaC | Codon optimized phaC in pUC57, AmpR | GenScript |
 pUC57-ZZ/ZZ | Codon optimized N-terminal Z domains in pUC57, AmpR | GenScript |
 pUC57-ZZ | Codon optimized C-terminal Z domains in pUC57, AmpR | GenScript |
 pMM1522 | Shuttle vector for cloning in E. coli (AmpR) and expression under xylose control in B. megaterium (TetR) | [34] |
 pMM-AB | Codon optimized phaA and phaB cloned BglII-BamHI and BamHI-KpnI, respectively, into pMM1522 | This study |
 pMM-CAB | Codon optimized phaC cloned BsrGI-BglII into pMM-AB | This study |
 pMM-ZZCAB | ZZ/ZZC-ZZ cloned BsrGI-BglII into pMM-CAB, replacing phaC | This study |
 pPT7 | Shuttle vector for cloning in E. coli (AmpR) and expression in B. megaterium (TetR). T7 promoter and terminator inserted to give PT7-MCS-Stop-TT7 | [33] |
 pPT7-AB | phaAB fragment from pMM-AB cloned BglII-KpnI into pPT7 | This study |
 pPT7-CAB | phaCAB fragment from pMM-CAB cloned BsrGI-KpnI into pPT7 | This study |
 pPT7-ZZCAB | ZZ/ZZC-ZZ cloned BsrGI-BglII into pPT7-CAB, replacing phaC | This study |
 pT7RNAP | Helper plasmid bearing T7 RNA polymerase under control of xylA promoter (CmR) | [33] |
 p1623hp (=p3STOP1623hp) | Shuttle vector for cloning in E. coli (AmpR) and expression under xylose control in B. megaterium (TetR) | [35] |
 p1623-ZZCAB | ZZ/ZZCZZ-AB fragment from pMM-ZZCAB cloned BsrGI-KpnI into p1623hp | This study |
 pPT7-ZZCAB(-SS) | N-terminal ZZ/ZZ region PCR amplified (without SS) from pPT7-ZZCAB and re-inserted into pPT7-CAB; C-terminal Z domains sucloned Bsu361-KpnI from pPT7-ZZCAB | This study |
 pMM1522PacI | G-block fragment ‘SalI-part of xylR-PxylA-PacI-SpeI’ inserted SalI-SpeI into pMM1522 | This study |
 pMM-ZZCAB(-SS) | ZZ/ZZC-ZZ-AB fragment subcloned PacI-KpnI from pPT7-ZCAB(-SS) to give pMM-ZCAB(-SS) | This study |
 pUC18 | E. coli cloning vector, AmpR. No origin of replication for B. megaterium | Thermo scientific |
 pMK-RQ-Cm | Gene synthesis of chloramphenicol resistance gene, flanked by FRT sites (FRT-CmR-FRT) in pMK-RQ, KanR | GeneArt |
 pUC18-FRT = pFRT | FRT sites (amplified from pMK-RQ-Cm (synthesis of FRT-CmR-FRT)) cloned HindIII-FRT-BamHI-FRT-KpnI into pUC18 | This study |
 pFRT-Cm | CmR gene PCR amplified from pMK-RQ-Cm with primers Cm-Fpsc and Cm-Rpsc (introducing SacI sites) and cloned SacI into pFRT | This study |
 pFRT-Cm-spoIIE | 500 nt fragment of B. megaterium spoIIE gene PCR amplified with primers SpoF-psc (NheI) and SpoR-psc (ApaI) and cloned NheI-ApaI into pFRT-Cm | This study |