TABLE 1.
Plasmids used in this work
| Plasmid name | Descriptiona | Reference or source |
|---|---|---|
| pMM1525 | Shuttle vector for xylose-inducible production and SPLipA-mediated secretion of target proteins; PxylA-splipA-mcs | 16 |
| pHIS1525 | Shuttle vector for xylose-inducible production and SPLipA-mediated secretion of C-terminally His6-tagged target proteins; PxylA-splipA-mcs-his6 | 16 |
| pC-HIS1622 | Shuttle vector for xylose-inducible intracellular production of C-terminally His6-tagged proteins; PxylA-mcs-his6-stop | 5 |
| pN-HIS-TEV1622 | Shuttle vector for xylose-inducible intracellular production of N-terminally His6-tagged proteins; PxylA-his6-tev-mcs-stop | 5 |
| pYYBm9 | Codon-optimized tfh-his6 cloned into BglII and EagI sites of pMM1525; PxylA-tfh-his6 | 35 |
| pADBm20 | Shuttle vector for xylose-inducible production and SPAsp-mediated secretion of target proteins; PxylA-spasp-mcs | 2 |
| pRBBm26 | Shuttle vector for xylose-inducible production and SPPac-mediated secretion of target proteins; PxylA-sppac-strep-xa-mcs | 3 |
| pRBBm34 | Shuttle vector for xylose-inducible intracellular production of Gfp proteins; PxylA-gfp | 4 |
| pSTOP1622 | Shuttle vector for xylose-inducible intracellular production of target proteins; PxylA-mcs-stop | 5 |
| p3STOP1622 | pSTOP1622 derivative encoding three stop codons for all possible reading frames downstream of mcs; PxylA-mcs-3stop | This work |
| p3STOP1623 | p3STOP1622 derivative containing an additional PacI restriction site; PxylA-mcs-3stop PacI | This work |
| p3STOP1624 | p3STOP1623 derivative containing an additional NheI restriction site; PxylA-mcs-3stop PacI-NheI | This work |
| p3STOP1622-gfp | gfp cloned into BglII and EagI sites of p3STOP1622; PxylA-gfp | This work |
| p3STOP1623-gfp | gfp cloned into BglII and EagI sites of p3STOP1623; PxylA-gfp PacI | This work |
| p3STOP1624-gfp | gfp cloned into BglII and EagI sites of p3STOP1624; PxylA-gfp PacI-NheI | This work |
| pKMBm1 | Annealed oligonucleotides −10+-for and −10+-rev inserted into p3STOP1624 (PacI-NheI); PxylA-(−10+)-mcs | This work |
| pSSBm40 | Annealed oligonucleotides utr+-for and utr+-rev inserted into p3STOP1624 (PacI-NheI); PxylA-(utr+)-mcs | This work |
| pSSBm44 | Annealed oligonucleotides rbs+-for and rbs+-rev inserted into p3STOP1624 (NheI-BsrGI); PxylA-(rbs+)-mcs | This work |
| pKMBm9 | gfp cloned into BglII and EagI sites of pKMBm1; PxylA-(−10+)-gfp | This work |
| pSSBm84 | p3STOP1624-gfp derivative encoding an optimized −35 region of PxylA; PxylA-(−35+)-gfp | This work |
| pSSBm46 | gfp cloned into BglII and EagI sites of pSSBm40; PxylA-(utr+)-gfp | This work |
| pSSBm50 | gfp cloned into BglII and EagI sites of pSSBm44; PxylA-(rbs+)-gfp | This work |
| pSSBm78 | pSSBm50 derivative encoding an optimized −35 region of PxylA; PxylA-(−35+rbs+)-gfp | This work |
| pSSBm74 | Optimized −10 region of PxylA cloned into pSSBm44; PxylA-(−10+rbs+)-mcs | This work |
| pSSBm76 | gfp cloned into BglII and EagI site of pSSBm74; PxylA-(−10+rbs+)-gfp | This work |
| pSSBm81 | Optimized −35 region of PxylA cloned into pSSBm76; PxylA-(−10+ −35+rbs+)-gfp | This work |
| pSSBm85 | pSSBm78 derivative lacking the NheI site; PxylA-(−35+rbs+)-gfp (ΔNheI) | This work |
| pKMBm4 | Annealed oligos utr+-for and utr+-rev inserted into pSSBm44 (PacI-NheI); PxylA-(utr+ rbs+)-mcs | This work |
| pKMBm10 | gfp cloned into BglII and EagI sites of pKMBm4; PxylA-(utr+ rbs+)-gfp | This work |
| p3STOP1623hp | mcs of p3STOP1624 inserted into pSSBm85; PxylA-(−35+rbs+)-mcs-3stop | This work |
| pC-HIS1623hp | mcs-his6 of pC-HIS1622 inserted into pSSBm85; PxylA-(−35+rbs+)-mcs-his6-stop | This work |
| pN-HIS-TEV1623hp | mcs-his6-tev of pN-HIS-TEV1622 inserted into pSSBm85; PxylA-(−35+rbs+)-his6-tev-mcs-stop | This work |
| pSSBm22 | spyngK cloned into BsrGI and BglII sites of pMM1525; PxylA-spyngK-mcs | This work |
| pSSBm23 | spvpr cloned into BsrGI and BglII sites of pMM1525; PxylA-spvpr-mcs | This work |
| pSSBm24 | spnprM cloned into BsrGI and BglII sites of pMM1525; PxylA-spnprM-mcs | This work |
| pSSBm25 | spyocH cloned into BsrGI and BglII sites of pMM1525; PxylA-spyocH-mcs | This work |
| pSSBm27 | Annealed oligonucleotides sp+yngK-for and sp+yngK-rev cloned into BsrGI and BglII sites of pMM1525; PxylA-sp+yngK-mcs | This work |
| pSSBm28 | tfh cloned into EagI and SpeI sites of pSSBm22; PxylA-spyngK-tfh | This work |
| pSSBm29 | tfh cloned into EagI and SpeI sites of pSSBm23; PxylA-spvpr-tfh | This work |
| pSSBm30 | tfh cloned into EagI and SpeI sites of pSSBm24; PxylA-spnprM-tfh | This work |
| pSSBm31 | tfh cloned into EagI and SpeI sites of pSSBm25; PxylA-spyocH-tfh | This work |
| pSSBm33 | tfh cloned into EagI and SpeI sites of pSSBm27; PxylA-sp+yngK-tfh | This work |
| pSSBm34 | spasp cloned into SpeI and XhoI sites of pSSBm28; PxylA-spasp-tfh | This work |
| pSSBm35 | sppac cloned into SpeI and XhoI sites of pSSBm28; PxylA-sppac-tfh | This work |
| pSSBm94 | spyngK-tfh subcloned from pSSBm28 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-spyngK-tfh | This work |
| pSSBm95 | spvpr-tfh subcloned from pSSBm29 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-spvpr-tfh | This work |
| pSSBm96 | spnprM-tfh subcloned from pSSBm30 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-spnprM-tfh | This work |
| pSSBm97 | spyocH-tfh subcloned from pSSBm31 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-spyocH-tfh | This work |
| pSSBm98 | sp+yngK-tfh subcloned from pSSBm33 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-sp+yngK-tfh | This work |
| pSSBm99 | spasp-tfh subcloned from pSSBm34 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-spasp-tfh | This work |
| pSSBm100 | sppac-tfh subcloned from pSSBm35 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-sppac-tfh | This work |
| pSSBm101 | splipA-tfh subcloned from pYYBm9 into BsrGI and PstI sites of p3STOP1623hp; PxylA-(−35+rbs+)-splipA-tfh | This work |
| pSPYngK-hp | mcs of pC-HIS1623hp inserted into SpeI and PstI sites of pSSBm94; PxylA-(−35+rbs+)-spyngK-mcs-his6 | This work |
| pSPVpr-hp | mcs of pC-HIS1623hp inserted into SpeI and PstI sites of pSSBm95; PxylA-(−35+rbs+)-spvpr-mcs-his6 | This work |
| pSPNprM-hp | mcs of pC-HIS1623hp inserted into SpeI and PstI sites of pSSBm96; PxylA-(−35+rbs+)-spnprM-mcs-his6 | This work |
| pSPYocH-hp | mcs of pC-HIS1623hp inserted into SpeI and PstI sites of pSSBm97; PxylA-(−35+rbs+)-spyocH-mcs-his6 | This work |
| pSP+YngK-hp | mcs of pC-HIS1623hp inserted into SpeI and PstI sites of pSSBm98; PxylA-(−35+rbs+)-sp+yngK-mcs-his6 | This work |
| pSPAsp-hp | mcs of pC-HIS1623hp inserted into SpeI and PstI sites of pSSBm99; PxylA-(−35+rbs+)-spasp-mcs-his6 | This work |
| pSPPac-hp | mcs of pC-HIS1623hp inserted into SpeI and PstI sites of pSSBm100; PxylA-(−35+rbs+)-sppac-mcs-his6 | This work |
| pSPLipA-hp | splipA-mcs of pC-HIS1525 inserted into BsrGI and PstI sites of pSSBm101; PxylA-(−35+rbs+)-splipA-mcs-his6 | This work |
a
A superscript plus sign indicates an optimized element as described in Materials and Methods.