Table 1.
Cloning parts, plasmids, strains and primers used in this study
| Cloning parts, plasmids, strains and primers | Description | Source/remark |
|---|---|---|
| RBS a | ||
| Rbs-O | AGGAGGACTAGCATG | [2] |
| Rbs-34 | ATTAAAGAGGAGAAAACTAGCATG | [15] |
| Rbs-30 | AAAGAGGAGAAAACTAGCATG | [15] |
| Rbs-H | TAGTGGAGGTACTAGCATG | RBS* from [15] |
| Rbs-C9 | AAAGGAGGTGATAGCATG | This study |
| Rbs-C10 | AAAGGAGGTGATTAGCATG | This study |
| Rbs-C11 | AAAGGAGGTGATCTAGCATG | This study |
| Promoter | ||
| Ptrc | TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACAC | [2] |
| 3 × Ptrc | TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACA | This study |
| 5 × Ptrc | TTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGTTGACAATTAATCATCCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACA | This study |
| Terminator | ||
| tt | Same as B0014 from http://www.partsregistry.org | [2] |
| Plasmids | ||
| pHKH001 | Integration vector disrupting slr0168 in Synechocystis genome with kanamycin resistance cassette a selection marker | [2] |
| pHKH_BB | Bio-brick compatible derivative of pHKH001 carrying the Ptrc promoter and tt-transcription terminator | This study |
| pHKH_rbsO_efe | Integration vector carrying efe under Ptrc promoter and Rbs-O | This study |
| pHKH_rbs34_efe | Integration vector carrying efe under Ptrc promoter and Rbs-34 | This study |
| pHKH_rbs30_efe | Integration vector carrying efe under Ptrc promoter and Rbs-30 | This study |
| pHKH_rbsH_efe | Integration vector carrying efe under Ptrc promoter and Rbs-H | This study |
| pHKH_rbsc9_efe | Integration vector carrying efe under Ptrc promoter and Rbs-C9 | This study |
| pHKH_rbsc10_efe | Integration vector carrying efe under Ptrc promoter and Rbs-C10 | This study |
| pHKH_rbsc11_efe | Integration vector carrying efe under Ptrc promoter and Rbs-C11 | This study |
| pHKH_3xPtrc_efe | Integration vector carrying efe under 3xPtrc promoter and Rbs-30 | This study |
| pHKH_5xPtrc_efe | Integration vector carrying efe under 5x Ptrc promoter and Rbs-30 | This study |
| pVZ_LS | A bio-brick compatible version of mobilizable broad host self-replication plasmid pVZ321 containing the RSF1010 replicon, and carrying the Ptrc promoter, Rbs30 and tt-transcription terminator | Plasmid pVZ321 from [42] |
| pVZ-efe | pVZ plasmid carrying efe gene under Ptrc promoter and rbs 30 | This study |
| pVZ_Nhis-efe | pVZ plasmid carrying N-terminal hexa-histidine tagged efe under promoter Ptrc and Rbs-30 | This study |
| Strains | ||
| Wild-type (WT) | Synechocystis sp. PCC6803, glucose tolerant, naturally transformable | Bhaya (Stanford) |
| VPV1 | WT, Slr0168::Ptrc-rbsO-efe-Kmr | This study |
| VPV2 | WT, Slr0168::Ptrc-rbs34-efe-Kmr | This study |
| VPV3 | WT, Slr0168::Ptrc-rbs30-efe-Kmr | This study |
| VPV4 | WT, Slr0168::Ptrc-rbsH-efe-Kmr | This study |
| VPV5 | WT, Slr0168::Ptrc-rbsc9-efe-Kmr | This study |
| VPV6 | WT, Slr0168::Ptrc-rbsc10-efe-Kmr | This study |
| VPV7 | WT, Slr0168::Ptrc-rbsc11-efe-Kmr | This study |
| VPV40 | WT, Slr0168::3xPtrc-rbsO-efe-Kmr | This study |
| VPV43 | WT, Slr0168::5xPtrc-rbsO-efe-Kmr | This study |
| SAW11 | Glycogen knock out strain | [36] |
| VPV55 | WT carrying plasmid pVZ _efe | This study |
| VPV56 | SAW11 carrying plasmid pVZ _efe | This study |
| VPV58 | SAW11 carrying plasmid pVZ_Nhis_efe | This study |
| VPV62 | WT carrying plasmid pVZ_Nhis_efe | This study |
| VPV65 | SAW11, Slr0168::Ptrc-rbs30-efe-Kmr | This study |
| Primers | ||
| efe-bb-fwd | GCGGAATTCGCGGCCGCTTCTAGAGGAGGACTAGCATGACCAACTTGCAAACCTTTGAAT | |
| efe-bb-r | GTACTGCAGCGGCCGCTACTAGTATTAGGAGCCGGTGGCGCG | |
| vpv-1-st-Hind-R | CTAGTAAAGCTTATCAATACTTTCCACCCC | |
| VPV-efe-Bamh1-f-noatg | CCATCACGGATCCACCAACTTGCAAACCTTTGAATTGC | |
| VPV-efe-st-Hind3-rev | TAATTAAGCTTATCAGGAGCCGGTGGCGCGGGTATCGG | |
| V-rbs30-efe-f | GACAGCTAGCATTAAAGAGGAGAAAACTAGCATGACCAACTTGCAAACC | |
| V-rbs34-efe-f | GACAGCTAGCAAAGAGGAGAAAACTAGCATGACCAACTTGCAAACC | |
| V-rbsC10-efe-f | GACAGCTAGCAAAGGAGGTGATTAGCATGACCAACTTGCAAACC | |
| V-rbsC11-efe-f | GACAGCTAGCAAAGGAGGTGATCTAGCATGACCAACTTGCAAACC | |
| V-rbsC9-efe-f | GACAGCTAGCAAAGGAGGTGATAGCATGACCAACTTGCAAACC | |
| V-rbsH-efe-f | GACAGCTAGCTAGTGGAGGTACTAGCATGACCAACTTGCAAACC | |
| v-bbs-pUC-r | CGGGATCCGATCCAATCTGCAGCGGCCGCTACTAGTA | |
| H1-SEG-VF | TGTCGCCGCTAAGTTAGACCGC | |
| H2-SEG-VR | CTGTGGGTAGTAAACTGGCAATGCC | |
| H1-SEQ-VF | CGGCAATGGTCCCAAAAT | |
| KanF-SEQ-VR | AGACGTTTCCCGTTGAAT | |
| pVZ321-SEQ-F | CGCAGGGCTTTATTGATT | |
| pVZ321-SEQ-R | CCCCCCCCACTCTATTGTA | |
| efe-n–H-f | CAGATATGACATATGCATCATCATCATCATCATACCAACTTGCAAACCTTTGAATTGC | |
| efe-ba-r | ACCTAGGTCAGTAGGATCCTTATTAGGAGCCGGTGGCGCGGGTATCG |
a RBS sequence underlined and start codon ATG is shown in bold