Table 1.
Bacterial strains, plasmids, and oligonucleotide primers used in this study
| Strain, plasmid, or primer | Relevant characteristic(s), description, or sequencea | Source or referenceb |
|---|---|---|
| Strains | ||
| E. coli DH5α | F−endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG ϕ80dlacZΔM15 Δ(lacZYA-argF)U169 hsdR17 (rK− mK+) λ− | Invitrogen |
| E. coli BW25113 | F− Δ(araD-araB)567 ΔlacZ4787(::rrnB-3) λ−rph-1 Δ(rhaD-rhaB)568 hsdR514 | CGSC |
| S. degradans 2-40 | Source of agarase genes and NABH | ATCC |
| Plasmids | ||
| pBAD vector | araBAD promoter (PBAD); Apr; pBR322 origin; araC; C-terminal polyhistidine tag; rrnB transcription termination region | 21 |
| pBAD-yfaL | pBAD carrying yfaL gene from E. coli K-12 | This study |
| pBAD-aga16B | pBAD carrying aga16B gene from S. degradans | This study |
| pBAD-mRFP1 | pBAD carrying mRFP1; expression in the cytoplasm | This study |
| pATLIC vector | Derivative of pBAD-yfaL; deletion of the passenger domain sequence of yfaL and addition of TEV protease recognition site | This study |
| pATLICsec vector | Derivative of pATLIC vector; addition of predicted cleavage site of YfaL | This study |
| pATLIC-NABH | pATLIC carrying NABH gene from S. degradans | This study |
| pATLIC-NABH | pATLICsec carrying NABH gene from S. degradans | This study |
| pATLIC*-mRFP1 | pATLIC carrying mRFP1 gene; lacking the translocator domain and expression in the periplasm | This study |
| pATLIC-mRFP1 | pATLIC carrying mRFP1 gene | This study |
| pATLIC-aga50A | pATLIC carrying aga50A gene from S. degradans | This study |
| pATLIC-aga16B | pATLIC carrying aga16B gene from S. degradans | This study |
| pATLIC-aga86C | pATLIC carrying aga86C gene from S. degradans | This study |
| pATLIC-aga50D | pATLIC carrying aga50D gene from S. degradans | This study |
| pATLIC-aga86E | pATLIC carrying aga86E gene from S. degradans | This study |
| pATLIC(m29-948)-mRFP1 | pATLIC carrying mRFP1 gene; deletion of 29-948 region of YfaL | This study |
| pATLIC(m29-785)-mRFP1 | pATLIC carrying mRFP1 gene; deletion of 29-785 region of YfaL | This study |
| pATLIC(m29-699)-mRFP1 | pATLIC carrying mRFP1 gene; deletion of 29-699 region of YfaL | This study |
| pATLIC(m29-692)-mRFP1 | pATLIC carrying mRFP1 gene; deletion of 29-695 region of YfaL | This study |
| Primers | ||
| yfaL_F | 5′-GAAGGAGATATAAGGATGCGGATTATCTTTCTACGCAAG-3′ | This study |
| yfaL_R | 5′-ATGATGGTGATGGTGCCATTTCACCGTCATCGACAAA-3′ | This study |
| aga16B_F | 5′-GAAGGAGATATAAGGATGGATTGGGACGGAATTCCTGTC-3′ | This study |
| aga16B_R | 5′-ATGATGGTGATGGTGACTGCCACCATTACCTGGGG-3′ | This study |
| mRFP1_F | 5′-GAAGGAGATATAAGGATGGCTTCCTCCGAAGACGTTATC-3′ | This study |
| mRFP1_R | 5′-ATGATGGTGATGGTGAGCACCGGTGGAGTGACG-3′ | This study |
| pATLIC948_F | 5′-CCCGGGCCAACGACCGAAAACCTTTACTTCCAGGCTTACCAGCCGGTGTTGAATGC-3′ | This study |
| pATLIC785_F | 5′-CCCGGGCCAACGACCGAAAACCTTTACTTCCAGCTTTCCAACGTGACGGTTAATGGC-3′ | This study |
| pATLIC699_F | 5′-GGCGGCGGGGGTGGGTCGTCACTGTGGGTTGGTGATGG-3′ | This study |
| pATLIC695_F | 5′-CACCACCACCATCATCACGGTGATGTTGCTGATATCCTTCCTTAT-3′ | This study |
| pATLIC29_R | 5′-GTTGGCCCGGGCGCGACACCGTTAGCAGAGAAAA-3′ | This study |
| mRFP129_F | 5′-CGGTGTCGCGCCCGCTTCCTCCGAAGACGTTATC-3′ | This study |
| mRFP1948_R | 5′-CGGTCGTTGGCCCAGCACCGGTGGAGTGACG-3′ | This study |
| mRFP1785_R | 5′-CGGTCGTTGGCCCAGCACCGGTGGAGTGACG-3′ | This study |
| mRFP1699_R | 5′-CCCGCCGCCCCCAGCACCGGTGGAGTGACG-3′ | This study |
| mRFP1692_R | 5′-GTGGTGGTGCCCAGCACCGGTGGAGTGACG-3′ | This study |
| mRFP1(stop)_R | 5′-CGGTCGTTGGCCCTTAAGCACCGGTGGAGTGAC-3′ | This study |
| NABH29-_F | 5′-CGGTGTCGCGCCCAGCGATTCAAAAGTAAATAAAAAATTG-3′ | This study |
| NABH785(tev)_R | 5′-CGGTCGTTGGCCCTACTGCTCCGGAATCGCCTG-3′ | This study |
| NAHB695(sec)_R | 5′-GTGGTGGTGCCCTACTGCTCCGGAATCGCCTG-3′ | This study |
| aga50A29_F | 5′-CGGTGTCGCGCCCGATAAAGACGAGCCGCAAGC-3′ | This study |
| aga50A785_R | 5′-CGGTCGTTGGCCCCTCGCCAAAACGTCGACTATAT-3′ | This study |
| aga16B29_F | 5′-CGGTGTCGCGCCCGATTGGGACGGAATTCCTGTC-3′ | This study |
| aga16B785_R | 5′-CGGTCGTTGGCCCGTTGCTAAGCGTGAACTTATCTAG-3′ | This study |
| aga86C29_F | 5′-CGGTGTCGCGCCCGGGGGCGGTAAATCCTCCAC-3′ | This study |
| aga86C785_R | 5′-CGGTCGTTGGCCCCTGCATAGGACGAGTAATTGAG-3′ | This study |
| aga50D29_F | 5′-CGGTGTCGCGCCCGGTGCAATTGGAGGTCTCGT-3′ | This study |
| aga50D785_R | 5′-CGGTCGTTGGCCCTTTGCTGCCTAGCCTTTCGG-3′ | This study |
| aga86E29_F | 5′-CGGTGTCGCGCCCGCCGATTATGTAATCGAAGCGG-3′ | This study |
| aga86E785_R | 5′-CGGTCGTTGGCCCTCTATTTGGCTCAGAAGTAAATTCC-3′ | This study |
a
The LIC sequences are underlined. Apr, ampicillin resistance.
b
CGSC, E. coli Genetic Stock Center; ATCC, American Type Culture Collection.