Table 4 Strains, plasmids, and primers used in this work
Strains, plasmids, or primers | Description | Source |
|---|---|---|
Strains | ||
 E. coli DH5α | F− (80d lacZ M15) (lacZYA-argF) U169 hsdR17(r− m+) recA1 endA1 relA1 deoR96 | Laboratory collection |
 E. coli BL21 (DE3) | F– ompT gal dcm lon hsdSB (rB− mB−) λ(DE3) | Laboratory collection |
 E. coli LGE2 | E. coli BL21 (DE3) harboring pET-lgk and pZBC | Ref. [17] |
 E. coli + lgk | E. coli BL21 (DE3) harboring pET-lgk | Ref. [17] |
 E. coli ΔxylF | Chromosome gene xylF-deleted E. coli BL21 (DE3) | This study |
 E. coli ΔkgtP | Chromosome gene kgtP-deleted E. coli BL21 (DE3) | This study |
 E. coli ΔxylF + lgk | E. coli ΔxylF harboring pET-lgk | This study |
 E. coli ΔkgtP + lgk | E. coli ΔkgtP harboring pET-lgk | This study |
 E. coli ΔxylF + lgk + xylF | E. coli ΔxylF harboring pET-xylF-lgk | This study |
 E. coli ΔkgtP + lgk + kgtP | E. coli ΔkgtP harboring pET-kgtP-lgk | This study |
Plasmids | ||
 pET-21a | ColE1 ori, f1 ori, Ampr, T7lac promoter | Laboratory collection |
 pET-lgk | pET-21a vector carrying a codon-optimized lgk gene derived from L. starkeyi | Laboratory collection |
 pCasPA | oriV, oriT, araC, araBAD promoter, T7 promoter, lac operator, λRed genes (Gam, Beta, Exo), SacB, Cas9, TetR, trfA | Purchased from Addgene repository |
 pACRISPR | pRO1600 oriV, ori, f1 ori, Ampr, T7 promoter, trc promoter, SP6 promoter, lac operator, gRNA scaffold, SacB | Purchased from Addgene repository |
 pACRISPR-sgRNAxylF | pACRISPR inserted with the sgRNA for gene xylF recognition | This study |
 pACRISPR-sgRNA-HRxylF | pACRISPR-sgRNAxylF inserted with the homologous arms for gene xylF deletion | This study |
 pACRISPR-sgRNAkgtP | pACRISPR inserted with the sgRNA for gene kgtP recognition | This study |
 pACRISPR-sgRNA-HRkgtP | pACRISPR-sgRNAxylF inserted with the homologous arms for gene kgtP deletion | This study |
 pET-xylF-lgk | pET-lgk vector carrying gene xylF | This study |
 pET-kgtP-lgk | pET-lgk vector carrying gene kgtP | This study |
Primers | ||
 F1 (for sgRNA of xylF) | 5′-GTGGGTCACATCGATCGGTGTCAGG-3′ | This study |
 R1 (for sgRNA of xylF) | 5′-AAACCCTGACACCGATCGATGTGAC-3′ | This study |
 F2 (for sgRNA of kgtP) | 5′-GTGGGTTCCTGATGCGCCCAATAGG-3′ | This study |
 R2 (for sgRNA of kgtP) | 5′-AAACCCTATTGGGCGCATCAGGAAC-3′ | This study |
 F3 (for left arm of xylF) | 5′-ACGTCTAGAGACAGCGTAGCGTCATCAGG-3′ | This study |
 R3 (for left arm of xylF) | 5′-ATCCTCGAGGCTAGCGCCTCCTGACACCGAT-3′ | This study |
 F4 (for right arm of xylF) | 5′-TAGGCTAGCGCAGCAACGTTGGTAAGCAG-3′ | This study |
 R4 (for right arm of xylF) | 5′-CAGCTCGAGTGACGGAATGCTAACGGGT-3′ | This study |
 F5 (for left arm of kgtP) | 5′-ACGTCTAGAGAATTTGCCTGGCGG-3′ | This study |
 R5 (for left arm of kgtP) | 5′-ATCCTCGAGGCTAGCGCGACGTGTATCA-3′ | This study |
 F6 (for right arm of kgtP) | 5′-TAGGCTAGCTGATGGCCGTGGTG-3′ | This study |
 R6 (for right arm of kgtP) | 5′-CAGCTCGAGAGGTTCGTAAACTCATCCG-3′ | This study |
 F7 (for restoration of xylF) | 5′-CATGAATTCTATATCTCCTTCTTAAAGTTAATTAC AGCTCGCTCTC-3′ | This study |
 R7 (for restoration of xylF) | 5′-CGCGGATCCACCATGAAAATAAAG-3′ | This study |
 F8 (for restoration of kgtP) | 5′-CATGAATTCTATATCTCCTTCTTAAAGTTAACTAA AGACGCATC-3′ | This study |
 R8 (for restoration of kgtP) | 5′-CGCGGATCCATGGCTGAAAGT-3′ | This study |