Team:Exeter/Parts
For our project we chose two enzymes:
- Perchlorate Reductase (Pcr)
- Chlorite Dismutase (Cld)
Basic Parts
Registry Number |
BioBricks |
Name |
Details |
Sequence Sources |
|---|---|---|---|---|
| BBa_K2695000 | Coding | A. suillum cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
Amino acid sequence translated from Dsui_0145 from Azospira suillum PS genome, GenBank CP003153 |
| BBa_K2695001 | Coding | A. oryzae cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
Genbank accession number: ADN79128.1 |
| BBa_K2695010 | Coding | D. aromatica cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction. Previously used by Leiden 2016 we have chosen to improve upon the activity of this enzyme.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
Genbank accession number: WP_011288310 |
| BBa_K2695002 | Coding | N. defluvii cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
GenBank accession number: ACE75544.1 |
| BBa_K2695003 | Coding | A. suillum pcrA sGFP | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The 28 amino acid signal peptide from A. suillum pcrA was inserted in front of sGFP (Pedelacq et. al., 2005). This was to determine whether the signal peptides from perchlorate reducing bacteria are functional in E. coli.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. | GFP accession number: PDB-2B3P |
| BBa_K2695004 | Coding | A. suillum |
Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The 22 amino acid signal peptide from A. suillum pcrC was inserted in front of sGFP (Pedelacq et. al., 2005). This was to determine whether the signal peptides from perchlorate reducing bacteria are functional in E. coli. For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
GFP accession number: PDB-2B3P |
| BBa_K2695005 | Coding | A. suillum cld sGFP | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The 31 amino acid signal peptide from A. suillum cld was inserted in front of sGFP (Pedelacq et. al., 2005). This was to determine whether the signal peptides from perchlorate reducing bacteria are functional in E. coli. For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
GFP accession number: PDB-2B3P |
| BBa_K2695006 | Coding | A. oryzae cld sGFP | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The 31 amino acid signal peptide from A. oryzae cld was inserted in front of sGFP (Pedelacq et. al., 2005). This was to determine whether the signal peptides from perchlorate reducing bacteria are functional in E. coli. For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
GFP accession number: PDB-2B3P |
| BBa_K2695007 | Coding | D. aromatica cld sGFP | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The 31 amino acid signal peptide from D. aromatica cld was inserted in front of sGFP (Pedelacq et. al., 2005). This was to determine whether the signal peptides from perchlorate reducing bacteria are functional in E. coli. For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
GFP accession number: PDB-2B3P |
| BBa_K2695008 | Coding | N. defluvii cld sGFP | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The 26 amino aicd signal peptide from N defluvii cld(Pedelacq et. al., 2005). This was to determine whether the signal peptides from perchlorate reducing bacteria are functional in E. coli. For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
GFP accession number: PDB-2B3P |
| BBa_K2695012 | Coding | pcrA | Synthesised with T7 promoter and the coding sequence for Azospira suillum pcrA. A His-tag inserted between the signal peptide and the enzyme sequence. PcrA forms a periplasmic heterodimer with PcrB and comprises the active complex of perchlorate reductase. (Youngblut et. al.,2016). | Amino acid sequence translated from Dsui_0149 from Azospira suillum PS Genome, GenBank CP003153 |
| BBa_K2695011 | Coding | pcrB, pcrC, pcrD | Synthesised with B0034 ribosome binding sites in front each of the coding sequences for Azospira suillum pcrB, pcrC, pcrD. PcrC has a Flag-tag inserted at the C terminal. PcrD has an HA-tag inserted at the C terminal. PcrB forms a periplasmic heterodimer with PcrA and comprises the active complex of perchlorate reductase. PcrC is a C-type cytochrome that connects the AB complex to the periplasmic membrane, allowing for electron shuffling. PcrD is predicted to be a molybdenum chaperone protein specific to assembling the pcrABC system. (Youngblut et. al.,2016) | Amino acid sequence translated from Dsui_0148, Dsui_0147 and Dsui_0146 from Azospira suillum PS Genome, GenBank CP003153 |
Part Collection
Teams may use the parts in this collection to allow their chassis to reduce chlorite to chloride and oxygen, which might be useful for soil detoxification or O2 production where chlorite or perchlorate is present. For perchlorate reduction, the enzymes below must be used along with Perchlorate Reductase (see above) as that enzyme reduces ClO4- to ClO2-, which is the substrate that chlorite dismutase uses.The 4 chlorite dismutase coding sequences in this collection are from 4 different species of bacteria. Exeter iGEM 2018 has shown, using a Western blot, that all coding sequences can be expressed in E. coli BL21(DE3). As the four enzymes have different kinetic parameters, future teams can choose which enzymes best suits their specific chassis. Choosing the right enzyme, with the right kinetic parameters will allow teams to balance metabolic flux of intermediate substrates through pathways whilst avoiding overburdening cells with unnecessary enzyme expression.
Registry Number |
BioBricks |
Name |
Details |
Sequence Source |
Part Type |
|---|---|---|---|---|---|
| BBa_K2695000 | Coding | A. suillum cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
Genebank accession number: AEV24567.1 | Basic |
| BBa_K2695001 | Coding | A. oryzae cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
Genebank accession number: ADN79128.1 | Basic |
| BBa_K2695002 | Coding | N. defluvii cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction. Previously used by Leiden 2016 we have chosen to improve upon the activity of this enzyme.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
Genebank accession number: WP_011288310 | Basic |
| BBa_K2695010 | Coding | D. aromatica cld | Submitted to iGEM as a basic part in pSB1C3 with a His-tag inserted between signal peptide and enzyme sequence. The enzyme catalyses the reduction of chlorite to chloride and oxygen. For our project it is the second step of our enzyme pathway to reduce perchlorate to oxygen. One of four chlorite dismutases used in this project, all with different predicted rates of reaction.
For use in the lab, modular cloning was used to add a T7 inducible promoter and B0015 terminator. |
Genebank accession number: ACE75544.1 | Basic |