TABLE 2.
Hybrid plasmids constructed in this studya
| Type of plasmid | Relevant characteristics | Structure |
|---|---|---|
| pCR2.1 derivatives | ||
| pCR5 | todC1(III from SacII site)-tecA2(5′ sequence to AatII site) | 0.2-kb fragment PCR amplified from pSTO4 template with primer pair prSTB70/71 |
| pCR6 | tecA1(I) | 0.6-kb fragment PCR amplified from pSTE7 template with primer pair prSTB72/73 |
| pCR7 | tecA1(II) | 0.5-kb fragment PCR amplified from pSTE7 template with primer pair prSTB74/76 |
| pCR8 | tecA1(III)-tecA2(5′ sequence to AatII site) | 0.5-kb fragment PCR amplified from pSTE7 template with primer pair prSTB77/71 |
| pCR9 | todC1 (I+IIABCD) (with NdeI site) | 0.9-kb fragment SOE-PCR-amplified from pSTO4 template with primer pairs prSTB72/73 and prSTB74/75 |
| pCR13 | tecA1(IIA) | 0.15-kb fragment PCR amplified from pSTE7 template with primer pair prSTB74/104 |
| pCR14 | tecA1(IIB)-todC1(IICD) | 0.15-kb fragment PCR amplified from pSTO4 template with primer pair prSTB114/75 |
| pCR15 | todC1(IIABC)-tecA1(IID) | 0.3-kb fragment PCR amplified from pSTO4 template with primer pair prSTB74/132 |
| pCR20 | todC1(IIB)-tecA1(IICDE) | 0.3-kb fragment PCR amplified from pSTE7 template with primer pair prSTB135/76 |
| pCR21 | tecA1(IIBCD) | 0.15-kb fragment PCR amplified from pSTE7 template with primer pair prSTB136/75 |
| pCR22 | tecA1(IIE) | 0.15-kb fragment PCR amplified from pSTE7 template with primer pair prSTB137/76 |
| pCR23 | tecA1(IIBCDE) | 0.3-kb fragment PCR amplified from pSTE7 template with primer pair prSTB136/76 |
| pBluescript KS II(+) derivatives | ||
| pSTE10.1 | pBluescript KS II(+) lacking part of its multiple cloning site | pBluescript KS II(+) religated after removal of EcoRV-Ecl136II fragment |
| pSTE11 | todC1-todC2 (5′ sequence to HindIII site) | 1.6-kb HindIII fragment of pSTO4 into HindIII site of pSTE10.1 |
| pSTE12 | todC1-tecA2 (5′ sequence to AatII site) | 0.2-kb SacII-AatII fragment of pCR5 into SacII-AatII site of pSTE11 |
| pSTE14 | todC1 (with NdeI site) | 0.8-kb SacI-BstEII fragment of pCR9 into SacI-BstEII site of pSTE12 |
| pSTE15 | tecA1(I)-todC1(II+III) | 0.6-kb SacI-NdeI fragment of pCR6 into SacI-NdeI site of pSTE14 |
| pSTE16 | todC1(I)-tecA1(II)-todC1(III) | 0.4-kb NdeI-RsrII fragment of pCR7 into NdeI-RsrII site of pSTE14 |
| pSTE17 | todC1(I+II)-tecA1(III) | 0.5-kb RsrII-AatII fragment of pCR8 into RsrII-AatII site of pSTE14 |
| pSTE21 | todC1(Phe211Leu) | Quikchange mutation of pSTE14 with primer pair prSTB105/124 |
| pSTE22 | todC1(Ser218Trp) | Quikchange mutation of pSTE14 with primer pair prSTB106/125 |
| pSTE23 | todC1(Met220Ala) | Quikchange mutation of pSTE14 with primer pair prSTB107/126 |
| pSTE24 | todC1(Gly224Ala) | Quikchange mutation of pSTE14 with primer pair prSTB108/127 |
| pSTE25 | todC1(Ser227Ala) | Quikchange mutation of pSTE14 with primer pair prSTB109/128 |
| pSTE26 | todC1(Phe211Ala) | Quikchange mutation of pSTE14 with primer pair prSTB110/129 |
| pSTE27 | todC1(Ser218Ala) | Quikchange mutation of pSTE14 with primer pair prSTB111/130 |
| pSTE28 | todC1(I)-tecA1(IIA)-todC1(IIBCDE+III) | 0.15-kb NdeI-StuI fragment of pCR13 into NdeI-StuI site of pSTE14 |
| pSTE30 | todC1(with NdeI site)-tecAaAbAcAd | 1.5-kb HindIII-AatII fragment of pSTE14 into HindIII-AatII site of pSTE7 |
| pSTE39 | todC1(I)-tecA1(IIAB)-todC1(IICDE+III) | 0.15-kb StuI-BstEII fragment of pCR14 into StuI-BstEII site of pSTE28 |
| pSTE41 | todC1(I)-tecA1(IIABCD)-todC1(IIE+III) | 0.15-kb StuI-BstEII fragment of pCR21 into StuI-BstEII site of pSTE28 |
| pSTE43 | todC1(I)-tecA1(IIAD)-todC1(IIBCE+III) | 0.15-kb StuI-BstEII fragment of pCR15 into StuI-BstEII site of pSTE28 |
| pSTE53 | todC1(I)-tecA1(II+III) | 0.4-kb NdeI-RsrII fragment of pSTE16 into NdeI-RsrII site of pSTE17 |
| pSTE61 | todC1(I)-tecA1(IIACDE)-todC1(IIB+III) | 0.3-kb StuI-RsrII fragment of pCR20 into StuI-RsrII site of pSTE28 |
| pSTE62 | todC1(I)-tecA1(IIAE)-todC1(IIBCD+III) | 0.15-kb BstEII-RsrII fragment of pCR22 into BstEII-RsrII site of pSTE28 |
| pSTE63 | todC1(I+IIACDE)-tecA1(IIB)-todC1(III) | 0.15-kb StuI-BstEII fragment of pCR14 into StuI-BstEII site of pSTE14 |
| pSTE64 | todC1(I+IIAE)-tecA1(IIBCD)-todC1(III) | 0.15-kb StuI-BstEII fragment of pCR21 into StuI-BstEII site of pSTE14 |
| pSTE65 | todC1(I+IIABCE)-tecA1(IID)-todC1(III) | 0.15-kb StuI-BstEII fragment of pCR15 into StuI-BstEII site of pSTE14 |
| pSTE66 | todC1(I+IIAB)-tecA1(IICDE)-todC1(III) | 0.3-kb StuI-RsrII fragment of pCR20 into StuI-RsrII site of pSTE14 |
| pSTE67 | todC1(I+IIABCD)-tecA1(IIE)-todC1(III) | 0.15-kb BstEII-RsrII fragment of pCR22 into BstEII-RsrII site of pSTE14 |
| pSTE68 | todC1(I+IIA)-tecA1(IIBCDE)-todC1(III) | 0.3-kb StuI-RsrII fragment of pCR23 into StuI-RsrII site of pSTE14 |
| Expressing dioxygenase genes | ||
| pSTO4 | todC1-C2BA | 4.1-kb EcoRI-BamHI fragment of pDTG601 into EcoRI-BamHI site of pBluescript II KS(+) |
| pSTE13 | todC1-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE12 into HindIII-AatII site of pSTE7 |
| pSTE18 | tecA1(I)-todC1(II+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE15 into HindIII-AatII site of pSTE7 |
| pSTE19 | todC1(I)-tecA1(II)-todC1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE16 into HindIII-AatII site of pSTE7 |
| pSTE20 | todC1(I+II)-tecA1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE17 into HindIII-AatII site of pSTE7 |
| pSTE29 | todC1(I)-tecA1(IIA)-todC1(IIBCDE+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE28 into HindIII-AatII site of pSTE7 |
| pSTE38 | tecA1(I+IIA)-todC1(IIBCDE)-tecA1(III)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE28 into NdeI-BstEII site of pSTE55 |
| pSTE40 | tecA1(I+IIAB)-todC1(IICDE)-tecA1(III)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE39 into NdeI-BstEII site of pSTE55 |
| pSTE42 | tecA1(I+IIABCD)-todC1(IIE)-tecA1(III)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE41 into NdeI-BstEII site of pSTE55 |
| pSTE46 | tecA1(I+IIAD)-todC1(IIBCE)-tecA1(III)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE43 into NdeI-BstEII site of pSTE55 |
| pSTE47 | todC1(I)-tecA1(IIAB)-todC1(IICDE+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE39 into HindIII-AatII site of pSTE7 |
| pSTE48 | todC1(I)-tecA1(IIABCD)-todC1(IIE+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE41 into HindIII-AatII site of pSTE7 |
| pSTE49 | todC1(I)-tecA1(IIAD)-todC1(IIBCE+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE43 into HindIII-AatII site of pSTE7 |
| pSTE52 | tecA1(I+II)-todC1(III)-tecA2A3A4 | 0.9-kb NdeI-AatII fragment of pSTE16 into NdeI-AatII site of pSTE18 |
| pSTE54 | todC1(I)-tecA1(II+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE53 into HindIII-AatII site of pSTE7 |
| pSTE55 | tecA1(I)-todC1(II)-tecA1(III)-tecA2A3A4 | 0.9-kb NdeI-AatII fragment of pSTE17 into NdeI-AatII site of pSTE18 |
| pSTE69 | todC1(I)-tecA1(IIACDE)-todC1(IIB+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE61 into HindIII-AatII site of pSTE7 |
| pSTE70 | todC1(I)-tecA1(IIAE)-todC1(IIBCD+III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE62 into HindIII-AatII site of pSTE7 |
| pSTE71 | todC1(I+IIACDE)-tecA1(IIB)-todC1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE63 into HindIII-AatII site of pSTE7 |
| pSTE72 | todC1(I+IIAE)-tecA1(IIBCD)-todC1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE64 into HindIII-AatII site of pSTE7 |
| pSTE73 | todC1(I+IIABCE)-tecA1(IID)-todC1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE65 into HindIII-AatII site of pSTE7 |
| pSTE74 | todC1(I+IIAB)-tecA1(IICDE)-todC1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE66 into HindIII-AatII site of pSTE7 |
| pSTE75 | todC1(I+IIABCD)-tecA1(IIE)-todC1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE67 into HindIII-AatII site of pSTE7 |
| pSTE76 | todC1(I+IIA)-tecA1(IIBCDE)-todC1(III)-tecA2A3A4 | 1.5-kb HindIII-AatII fragment of pSTE68 into HindIII-AatII site of pSTE7 |
| pSTE79 | todC1(Phe211Leu)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE21 into NdeI-BstEII site of pSTE30 |
| pSTE80 | todC1(Ser218Trp)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE22 into NdeI-BstEII site of pSTE30 |
| pSTE81 | todC1(Met220Ala)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE23 into NdeI-BstEII site of pSTE30 |
| pSTE82 | todC1(Gly224Ala)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE24 into NdeI-BstEII site of pSTE30 |
| pSTE83 | todC1(Ser227Ala)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE25 into NdeI-BstEII site of pSTE30 |
| pSTE84 | todC1(Phe211Ala)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE26 into NdeI-BstEII site of pSTE30 |
| pSTE85 | todC1(Ser218Ala)-tecA2A3A4 | 0.3-kb NdeI-BstEII fragment of pSTE27 into NdeI-BstEII site of pSTE30 |
| pSTE86 | todC1(Met220Ala)-todC2BA | 1.0-kb EcoRI-BspEI fragment of pSTE81 into EcoRI-BspEI site of pSTO4 |
a
The relevant gene regions correspond to those defined in Fig. 3.