U6PT design
As our team decided to clone part of the biosynthesis pathway of decursin starting from umbelliferone, the first enzyme needed was Umbelliferone-6 prenyltransferase (U6PT). Due to the insufficiency of information regarding the cloning of plant aromatic prenyltransferases in bacterial systems, we decided to expand our cloning efforts and work with six different U6PT genes; four of which are from a plant source, and two were designed by our team based on other plant prenyltransferases, for more information go to our Design page.
Our basic parts in this design include the six U6PTs we used. All of those enzymes mediate prenylation reactions in which dimethylallyl diphosphate (DMAPP) is the prenyl donor, and umbelliferone is the acceptor, yielding 7-demethylsuberosin (DMS). Moreover, each of them was part of a composite part, including our new P2A self-cleavage peptide sequence, optimized for E. coli , along with a mCherry reporter gene. The entire construct was planned to be cloned under the rhlR promoter.
The backbone used in this design was A133-rhlr-tdPP7-mCherry plasmid, which contains a rhlR promoter along with a rlhR regulator operon, which was gifted by Prof. Roee Amit’s lab.
As we were not able to get results on these enzymes due to time restrictions, we made sure to add their proper documentation from literature sources and we aim to update their pages with additional results once the registry opens again.
We have compiled all the new parts we added to the registry in the following table:
| Part name | Part number | Part type | Basic / Composite | Length |
|---|---|---|---|---|
| Petroselinum crispum prenyltransferase (PcPT) | BBa_K4179000 | Coding | Basic | 1212 |
| Pastinaca sativa prenyltransferase 1 (PsPT1) | BBa_K4179001 | Coding | Basic | 1215 |
| Ficus caricia prenyltransferase (FcPT1) | BBa_K4179002 | Coding | Basic | 1284 |
| Umbelliferone dimethylallyltransferase (UDT) | BBa_K4179007 | Coding | Basic | 1200 |
| PT1a with the donor docking domain switched to the domain of PT1L | BBa_K4179011 | Coding | Basic | 1218 |
| PT1a with the donor docking domain switched to the domain of G4DT | BBa_K4179012 | Coding | Basic | 1218 |
| P2A self-cleaving peptide - Optimized for E. coli | BBa_K4179005 | DNA | Basic | 66 |
| PcPT under Rhlr promoter + P2A + mCherry reporter | BBa_K4179003 | Composite | Composite | 2243 |
| PsPT1 under Rhlr promoter + P2A + mCherry reporter | BBa_K4179008 | Composite | Composite | 2246 |
| FcPT1 under Rhlr promoter + P2A + mCherry reporter | BBa_K4179009 | Composite | Composite | 2315 |
| UDT under Rhlr promoter + P2A + mCherry reporter | BBa_K4179010 | Composite | Composite | 2231 |
| ChimeraI under Rhlr promoter + P2A + mCherry reporter | BBa_K4179013 | Composite | Composite | 2249 |
| ChimeraII under Rhlr promoter + P2A + mCherry reporter | BBa_K4179014 | Composite | Composite | 2249 |
Xim design
The next step in the decursin biosynthesis pathway is the conversion of DMS to decursinol. This reaction is catalyzed by the pair of enzymes, XimD and XimE.
Our team was interested in the optimal ratio between the two enzymes, and after multiple iterations of this design based on our Design-Build-Test approach, the final genetic system of the Xim design contains XimE under a constitutive pT7 promoter, and XimD under a LacI-regulated pT7 promoter.
The new parts added to registry contain the two Xim enzymes, and all other parts in this system were already in the iGEM registry.
The backbone used in this design is #172654 from AddGene.
The following list carries our new parts in the Xim design:
| Part name | Part number | Part type | Basic / Composite | Length |
|---|---|---|---|---|
| flavin-dependent monooxygenase - XimD | BBa_K4179016 | Coding | Basic | 1422 |
| SnoaL-like cyclase - XimE | BBa_K4179018 | Coding | Basic | 375 |
| XimD + XimE under T7 promoters + LacI + Lac operator | BBa_K4179019 | Composite | Composite | 3288 |
Regulation circuit of XimE
OraCell
This design is dedicated to our new measuring tool OraCell. A system based on Luciferase assay, which takes advantage of the existing Hippo pathway in mammalian cells, and can be used to detect and quantify not only decursin, but multiple other metabolites. For more details about this system visit our OraCell page.
The system is comprised of two main segments; the first contains the Hippo pathway components that are affected by decursin, those include eight GTIIC motifs with a high affinity for specific transcription factors which cause down-expression of the Luciferase gene when decursin is present in the system. The second includes components for blasticidine resistance gene BlastR, which is required for transfection of mammalian cells.
Most parts in this system already exist in the iGEM registry, however, we did add two basic parts that were missing. In addition, we added the composite part of the whole system.
Results are shown on the OraCell page, as well as the composite part's registry page.
The list below holds all new parts our team added for OraCell design:
| Part name | Part number | Part type | Basic / Composite | Length |
|---|---|---|---|---|
| 8xGTIIC | BBa_K4179017 | Protein domain | Basic | 134 |
| cTNT promoter | BBa_K4179021 | Regulatory | Basic | 134 |
| Luc_Blast_Oracell | BBa_K4179020 | Composite | Composite | 3291 |