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We are happy to share all our designed DNA parts as well as our experience using them with the iGEM community. Below you find parts for perchlorate reductase and chlorite dismutase expression in E. coli and B. subtilis.
| Part name | iGEM Registry Number | Description |
|---|---|---|
| RBS_pcrA | BBa_25T2YTZ1 | RBS variation of the Shine-Dalgarno sequence [1]. Additional bases were inserted between the RBS and the start codon to maintain an optimal spacing of 7–9 nucleotides for Bacillus subtilis [2]. |
| pcrA_B.sub | BBa_25IPSE0T | Encodes for perchlorate reductase α subunit. Responsible for reducing perchlorate (ClO4-) to chlorite (ClO2-) during anaerobic respiration [3]. Optimized for expression in B. subtilis. |
| RBS_pcrB | BBa_25SV4PZ2 | RBS variation of the Shine-Dalgarno sequence [1]. Additional bases were inserted between the RBS and the start codon to maintain an optimal spacing of 7–9 nucleotides for Bacillus subtilis [2]. |
| pcrB_B.sub | BBa_25489COQ | Encodes perchlorate reductase β subunit. Transfers electrons to the catalytic α subunit (PcrA) [4]. Optimized for expression in B. subtilis. |
| RBS_pcrC | BBa_2582Q7YN | RBS variation of the Shine-Dalgarno sequence [1]. Additional bases were inserted between the RBS and the start codon to maintain an optimal spacing of 7–9 nucleotides for Bacillus subtilis [2]. |
| pcrC_B.sub | BBa_25Y6C7QD | Encodes a c-type cytochrome, functions as an electron shuttle, transferring electrons from the quinone pool to the PcrAB catalytic complex during perchlorate reduction [4]. Optimized for expression in B. subtilis |
| RBS_pcrD | BBa_256VT3PD | RBS variation of the Shine-Dalgarno sequence [1]. Additional bases were inserted between the RBS and the start codon to maintain an optimal spacing of 7–9 nucleotides for Bacillus subtilis [2]. |
| pcrD_B.sub | BBa_25AUICYM | Encodes a molybdenum chaperone protein involved in the assembly of PcrA [4]. Optimized for expression in B. Subtilis. |
| RBS_cld | BBa_25ONHJ8I | RBS variation of the Shine-Dalgarno sequence [1]. Additional bases were inserted between the RBS and the start codon to maintain an optimal spacing of 7–9 nucleotides for Bacillus subtilis [2]. |
| cld_B.sub | BBa_25FP4KBG | Encodes a heme b-dependent enzyme that catalyzes the decomposition of toxic chlorite into harmless chloride and molecular oxygen [5]. Optimized for expression in B. subtilis. |
| p43_promoter | BBa_K143013 | Constitutive promoter found in Bacillus subtilis. Registered by iGEM Imperial College London 2008. |
| Ao_pcrA | BBa_25TL8PZY | Encodes for perchlorate reductase α subunit. Responsible for reducing perchlorate (ClO₄⁻) to chlorite (ClO₂⁻) during anaerobic respiration [3]. Obtained from Azospira oryzae GR-1 genome. Used for expression in E. coli. |
| Ao_pcrB | BBa_252ZK706 | Encodes perchlorate reductase β subunit. Transfers electrons to the α subunit (PcrA) [4]. Obtained from Azospira oryzae GR-1 genome. Used for expression in E. coli. |
| Ao_pcrD | BBa_25RLU06M | Encodes a molybdenum chaperone protein involved in the assembly of the PcrABC enzyme complex [4]. Obtained from Azospira oryzae GR-1 genome. Used for expression in E. coli. |
Chlorite dismutase (A. oryzae GR-1)  |
BBa_25VZ3X3W | Encodes a heme b-dependent enzyme that catalyzes the decomposition of toxic chlorite into harmless chloride and molecular oxygen [5]. Obtained from cld_B.sub. Exhibits N-terminal hexahistidine tag for purification. |
| cld_monomers_E.coli | Design 1: BBa_25JSFQN5 Design 2: BBa_25K3UY69 Design 3: BBa_2530V0VQ Design 4: BBa_25DOV1JJ Cld-GFP: BBa_2550CDC9 |
Chlorite dismutase variants, which exhibit mutations to change oligomeric structure. Optimized for expression in E. coli. |
| His_terminator | BBa_K3143688 | Terminator of the pcrABD operon for E. coli expression. Registered by Meng Fankang 2019. |
| Ao_pcrB_RBS | BBa_259DN028 | RBS of pcrB from Azospira oryzae GR-1 genome. Used for expression of pcrB in E. coli. |
| Ao_pcrC_RBS | BBa_25IMVFF4 | RBS of pcrC from Azospira oryzae GR-1 genome. Used for expression of pcrD in E. coli. |
| Ptac promoter | BBa_25IVX5PZ | Ptac is a hybrid promoter derived from the trp and lac promoters of E. coli. It is regulated by the LacI repressor and can be induced by isopropyl β-D-1-thiogalactopyranoside (IPTG). Registered by Yun-Lu Cheng 2025. |
| RBS | BBa_25CUH7KV | RBS for expression of pcrA in E. coli. |
| pcrA_Y165H | BBa_25B9XFYS | Encodes the molybdopterin containing subunit of the perchlorate reductase reducing protein complex PcrAB [3] tagged with Strep-Tag II N-terminally and mutated at position Y164 to histidine. Supposed to reduce chlorate and perchlorate if expressed with pcrB and pcrD of the perchlorate reductase operon. |
| pcrA_W461H | BBa_25GBU5P1 | Encodes the molybdopterin containing subunit of the perchlorate reductase reducing protein complex PcrAB [3] tagged with Strep-Tag II N-terminally and mutated at position W461 to histidine. Supposed to reduce chlorate and perchlorate if expressed with pcrB and pcrD of the perchlorate reductase operon. |
| Part name | iGEM Registry Number | Description |
|---|---|---|
| pcrABCD_B.sub | BBa_25PX1P56 | Full perchlorate reductase operon under the control of the p43 promoter. Optimized for expression in B. subtilis. |
| cld_full_construct_B.sub | BBa_25ENBW8R | Chlorite dismutase (cld) transcriptional unit. Expression is regulated by the p43 promoter and transcription is terminated by the tbcS terminator. Optimized for expression in B. subtilis strain 168 |
| pcrABD_E.coli |
BBa_254G21JZ | Perchlorate reductase operon for expression in E. coli BL21. |
| pcrABD_Y165H_E.coli | BBa_25XWORJJ | Perchlorate reductase operon with mutation in PcrA Y165H for expression in E. coli BL21. |
| pcrABD_W461H_E.coli | BBa_25KJQYQ9 | Perchlorate reductase operon with mutation in PcrA W461H for expression in E. coli BL21. |
| Backbone | Function |
|---|---|
| pMK4 | Shuttle vector compatible with E. coli and B. subtilis [7]. Used as the backbone for pcrABCD operon and cld expression system. Provided by AG Simon (TU Darmstadt). |
| pUPD | Domestication vector used for GoldenBraid cloning [6]. Provided by AG Warzecha (TU Darmstadt). |
| pDGB2-αE1 | GoldenBraid alpha vector [6] deprived of agrobacterial features such as ori and resistance markers. Used for protein expression in E. coli. Provided by AG Warzecha (TU Darmstadt). |
| pQE | Expression vector for overexpression of N-terminally His-tagged proteins in E. coli BL21. Used for cld expression. Provided by AG Warzecha (TU Darmstadt). |
[1] J.-D. Wen, S.-T. Kuo, and H.-H. D. Chou, "The diversity of Shine-Dalgarno sequences sheds light on the evolution of translation initiation," RNA Biology, vol. 18, no. 11, pp. 1489–1500, 2021, doi: 10.1080/15476286.2020.1861406.
[2] K. Volkenborn, L. Kuschmierz, N. Benz, P. Lenz, A. Knapp, and K.-E. Jaeger, "The length of ribosomal binding site spacer sequence controls the production yield for intracellular and secreted proteins by Bacillus subtilis," Microbial Cell Factories, vol. 19, no. 1, p. 154, 2020, doi: 10.1186/s12934-020-01404-2.
[3] M. Nozawa-Inoue, M. Jien, N. S. Hamilton, V. Stewart, K. M. Scow, and K. R. Hristova, "Quantitative detection of perchlorate-reducing bacteria by real-time PCR targeting the perchlorate reductase gene," Applied and Environmental Microbiology, vol. 74, no. 6, pp. 1941–1944, 2008, doi: 10.1128/AEM.01658-07.
[4] K. S. Bender, C. Shang, R. Chakraborty, S. M. Belchik, J. D. Coates, and L. A. Achenbach, "Identification, characterization, and classification of genes encoding perchlorate reductase," Journal of Bacteriology, vol. 187, no. 15, pp. 5090–5096, 2005, doi: 10.1128/JB.187.15.5090-5096.2005.
[5] A. Q. Lee, B. R. Streit, M. J. Zdilla, M. M. Abu-Omar, and J. L. Dubois, "Mechanism of and exquisite selectivity for O-O bond formation by the heme-dependent chlorite dismutase," Proceedings of the National Academy of Sciences of the United States of America, vol. 105, no. 41, pp. 15654–15659, 2008, doi: 10.1073/pnas.0804279105.
[6] A. Sarrion-Perdigones et al., "GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology," Plant Physiology, vol. 162, no. 3, pp. 1618–1631, 2013, doi: 10.1104/pp.113.217661.
[7] M. A. Sullivan, R. E. Yasbin, and F. E. Young, "New shuttle vectors for Bacillus subtilis and Escherichia coli which allow rapid detection of inserted fragments," Gene, vol. 29, no. 1–2, pp. 21–26, 1984, doi: 10.1016/0378-1119(84)90161-6.