Table 1.
Strains, plasmids, and primers
| Strain, plasmid, or primer | Derivation, relevant characteristics, or sequences (5′→3′)a | Source, reference(s), or gene |
|---|---|---|
| Synechocystis sp. strains | ||
| PCC 6803 | Wild type, a glucose-tolerant strain | J. Zhao, Beijing University/Institute of Hydrobiology |
| DRHB818b | Kmr, ggpS::C.K, Synechocystis 6803 transformed with pHB818 | This study |
| DRHB2548 | Cmr Emr, rbp1::C.CE2, Synechocystis 6803 transformed with pHB2548 | This study |
| DRHB2548/DRHB2788 | Cmr Emr Kmr, rbp1::C.CE2 complemented with the wild-type rbp1, mutant DRHB2548 transformed with pHB2788 | This study |
| DRHB2549 | Kmr, rbp2::C.K, Synechocystis 6803 transformed with pHB2549 | This study |
| DRHB2791 | Smr Spr, PrbcL-ccr2 in addition to the indigenous ccr2; Synechocystis 6803 transformed with pHB2791 to introduce PrbcL-ccr2 into a neutral platform (6, 24) of the genome | This study |
| DRHB3288 | Smr Spr, PrbcL-rbp1 in addition to the indigenous rbp1; Synechocystis 6803 transformed with pHB3288 to introduce PrbcL-rbp1 into a neutral platform (6, 24) of the genome | This study |
| Plasmidsc | ||
| pHB796 | Apr; the PCR fragment containing ggpS, amplified with primers sll1566-1 and sll1566-2, cloned into pMD18-T | This study |
| pHB818 | Apr Kmr; the C.K cassette excised with PvuII from pRL446, blunted with T4 DNA polymerase, cloned into the BalI site of ggpS within pHB796 | This study |
| pHB2489 | Apr; the PCR fragment containing rbp1 coding region, amplified with primers sll0517-a1 and sll0517-a2, cloned into pMD18-T | This study |
| pHB2502 | Apr; the PCR fragment overlapping the 5′ end and upstream sequence of rbp1, amplified with primers sll0517-k1 and sll0517-k2, cloned into pMD18-T | This study |
| pHB2503 | Apr; the PCR fragment overlapping the 3′ end and downstream sequence of rbp1, amplified with primers sll0517-k3 and sll0517-k4, cloned into pMD18-T | This study |
| pHB2504 | Apr; the PCR fragment overlapping the 5′ end and upstream sequence of rbp2, amplified with primers ssr1480-k1 and ssr1480-k2, cloned into pMD18-T | This study |
| pHB2505 | Apr; the PCR fragment overlapping the 3′ end and downstream sequence of rbp2, amplified with primers ssr1480-k3 and ssr1480-k4, cloned into pMD18-T | This study |
| pHB2535 | Apr Cmr Emr; the C.CE2 cassette excised with BamHI from pRL598, blunted with T4 DNA polymerase, cloned into the blunted SalI site of pHB2503 | This study |
| pHB2536 | Apr Kmr; the C.K cassette excised with BamHI from pRL446, blunted with T4 DNA polymerase, cloned into the blunted SalI site of pHB2505 | This study |
| pHB2548 | Apr Cmr Emr; the DNA fragment containing C.CE2 and the downstream sequence of rbp1 excised with PstI and SmaI from pHB2535, blunted with T4 DNA polymerase, cloned into the SmaI site of pHB2502, with C.CE2 positioned between the flanking sequences of rbp1 | This study |
| pHB2549 | Apr Kmr; the DNA fragment containing C.K and the downstream sequence of rbp2 was excised with SmaI and PstI from pHB2536, blunted with T4 DNA polymerase, cloned into the SmaI site of pHB2504, with C.K positioned between the flanking sequences of rbp2 | This study |
| pHB2560 | Kmr; the DNA fragment excised with NcoI and XhoI from pHB2489, cloned into pET41a, for production of Synechocystis Rbp1 with six-His tag in Escherichia coli | This study |
| pHB2706 | Apr; the PCR fragment containing ccr2, amplified with primers ccr2-e1 and ccr2-e2, cloned into pMD18-T | This study |
| pHB2739 | Apr; the PCR fragment containing the rbcL promoter amplified with primers PrbcL-1 and PrbcL-6, cloned into pMD18-T | This study |
| pHB2759 | Apr Spr; Ω cassette excised with DraI from pRL57, cloned into SalI-cut and T4 DNA polymerase-blunted pHB2739 | This study |
| pHB2761 | Apr; the PCR fragment containing rbp1, amplified with primers sll0517-k1 and sll0517-a4, cloned into pMD18-T | This study |
| pHB2768 | Apr Kmr; the C.K cassette excised with BamHI from pRL446, cloned into the BamHI site of pHB2761 | This study |
| pHB2770 | Apr Spr; Ω-PrbcL excised with XbaI/PstI from pHB2759, blunted with T4 DNA polymerase, cloned into XbaI-cut and T4 DNA polymerase-blunted pHB2706 | This study |
| pHB2788 | Apr Kmr; the fragment containing the C.K cassette and rbp1 excised with PvuII from pHB2768, cloned between the blunted EcoRI sites of pKW1188 | This study |
| pHB2791 | Apr Spr; Ω-PrbcL-ccr2 excised with SmaI and HincII from pHB2770, cloned between the blunted EcoRI sites of pKW1188 | This study |
| pHB3208 | Apr; the PCR fragment containing the open reading frame of rbp1, amplified with primers sll0517-oe1 and sll0517-a4, cloned into pMD18-T | This study |
| pHB3284 | Apr Smr Spr; the Ω cassette excised with DraI from pRL57, cloned into the blunted SacI site of pHB2739, downstream of the cloned PrbcL | This study |
| pHB3287 | Apr Smr Spr; the DNA fragment containing PrbcL and Ω cassette, excised with PvuII and SalI from pHB3284 and blunted with T4 DNA polymerase, cloned between the blunted EcoRI sites of pKW1188, replacing the kanamycin resistance gene | This study |
| pHB3288 | Apr Smr Spr; the DNA fragment containing the rbp1 coding region, excised with SalI and XbaI from pHB3208 and blunted with T4 DNA polymerase, cloned into SmaI-cut and dephosphorylated pHB3287, located between PrbcL and Ω cassette, oriented as PrbcL | This study |
| pET21b | Apr; overexpression vector | Novagen, EMD Chemicals Inc. |
| pET41a | Kmr; overexpression vector | Novagen |
| pKW1188 | Apr Kmr; a plasmid bearing a neutral integrative platform for Synechocystis 6803 | 6, 24 |
| pMD18-T | Apr; cloning vector | Takara, Japan |
| pRL57 | Kmr Smr Spr; a pDU1-based plasmid containing the spectinomycin resistance cassette Ω | 5 |
| pRL446 | Apr Kmr; a plasmid containing the kanamycin resistance cassette C.K | NCBI GenBank accession no. EU346690 |
| pRL598 | Apr Cmr Emr; a plasmid containing the chloramphenicol and erythromycin resistance cassette C.CE2 | 5 |
| Primers (5′→3′) | ||
| ccr2-e1 | GGCTGTTACTCCAGACCCA | ccr2 |
| ccr2-e2 | AGCAAGACAACAATGGACAGGA | |
| sll1566-1 | CCTGGTCAATGGATTCGTCC | ggpS |
| sll1566-2 | GTGAGCCCTACGACGAAGT | |
| gvpAC-1 | C(C/T)TACCTCAAATATGCTGAAGC | gvpA-gvpC intergenic sequence |
| gvpAC-2 | TGCCTGTTCTTGCGCTTGT | |
| PrbcL-1 | CCGATGAAGTGGTGGAGCA | rbcL |
| PrbcL-6 | GGTCAGTCCTCCATAAACATTG | |
| sll0517-a1 | ACCATGGTGTCAATTTATGTAGGCAACCTGTCC | rbp1 |
| sll0517-a2 | TTCTCGAGGTAGCGGCTACCACCATAGCT | |
| sll0517-a3d | TCTCATATGTCAATTTATGTAGGCAACCTGTCC | |
| sll0517-a4d | TTTCTCGAGTGGTGGAACGACGGCGAA | |
| sll0517-k1 | GTAGAAACGGGTACTGGTCATG | |
| sll0517-k2 | GTTGCCTACATAAATTGACATGGATT | |
| sll0517-k3 | TTCCTTTGGTGGCGGTCGT | |
| sll0517-k4 | CTCCTCCGAATCCTTGCGAA | |
| sll0517-oe1 | GTTTTTGGAGAAAATCCATGTCAA | |
| ssr1480-a3d | TTTCATATGTCCATTTATGTCGGGAACCTTTCTT | rbp2 |
| ssr1480-a4d | TTTCTCGAGGACTCAAACACCTTCCCTTCTACAA | |
| ssr1480-k1 | CGGCTACTGTGAATCTTTGGA | |
| ssr1480-k2 | GGTTCCCGACATAAATGGACA | |
| ssr1480-k3 | AAAGCAAGACCGAGAACCCCT | |
| ssr1480-k4 | ACTCCCTTCAAATCTGGCTTCA | |
| rnpB-1d | GTTAGGGAGGGAGTTGCGG | rnpB |
| rnpB-2d | AAGAGAGTTAGTCGTAAGCCG |
a
Abbreviations: Ap, ampicillin; Cm, chloramphenicol; Em, erythromycin; Km, kanamycin; Sm, streptomycin; Sp, spectinomycin.
b
DRHBxxxx refers to a product of double homologous recombination between plasmid pHBxxxx and the Synechocystis sp. genome.
c
Unless stated otherwise, the template for PCRs was Synechocystis sp. genomic DNA.
d
These primers were used to generate probes for Northern blot hybridizations.