We aim to develop the parts for 5-HTP production and enhancement in the SERENE project.
To produce 5-HTP, we expressed the monomeric human Tryptophan Hydroxylase 1 (m-hTPH1) protein, which requires the cofactor tetrahydrobiopterin (BH4). To establish a BH4 regeneration system, we also expressed the human pterin-4a-carbinol-amine dehydratase (hPCBD1) protein and human q-dihydropteridine reductase (hQDPR) protein.
To enhance 5-HTP production, we applied rolling circle replication (RCR) to generate DNA scaffolds to cluster m-TPH1, hPCBD1, and hQDPR, each fused with different zinc finger protein binding domains. To regulate DNA scaffold formation, we applied protocatechuate (PCA) induced promoter pPCA to control the expression of RepA, the replication initiation protein in RCR.
Together, we believed that 5-HTP production system and the part of the PCA regulating system would contribute to the iGEM teams in the future.
| Part number | Name | Description | Length |
|---|---|---|---|
| BBa_K4674000 | mGreeen Lantern (mGL) | mGL protein is a green fluorescent protein with excitation and emission peaks at 503 and 514 nm, respectively. It differs from EGFP by 21 mutations. It possesses a higher cellular brightness (630% higher than EGFP) and matures rapidly (207% faster than EGFP). |
720 bp |
| BBa_K4674007 | RCORI-105 | The motif RCORI105 is a 105 bp sequence at the 3' terminus of the double-stranded origin of R-plasmid pC194. RCORI-105 serves as the start point of RepA mediated circular ssDNA synthesis in rolling circle replication. |
105 bp |
| BBa_K4674008 | RCORI-65 | The motif RCORI-65 is 65 bp sequence at the 5' terminus of double-stranded origin of R-plasmid pC194. RCORI-65 serves as the stop point of RepA mediated circular ssDNA synthesis in rolling circle replication. |
65 bp |
| BBa_K4674002 | RepA | The RepA is a replication initiator protein that initiate the process of rolling circle replication (RCR) of the R-plasmid, pC194. RepA binds to the double-stranded origin (DSO) and creates nick on one of the DNA strand. The DNA polymerase use unnicked strand as template to elongate the nicked strand. Finally, the elongated nicked strand is completely replaced by the newly synthesised strand, and the replication initiator enzyme will then ligate the elongated nicked strand into a circular DNA. |
690 bp |
| BBa_K3395008 | pLacIQ promoter | pLacIQ works as a constitutive promoter, and transcription will be higher with its help. | 54 bp |
| BBa_K1679038 | RiboJ insulator | RiboJ is a functional RNA that includes a self-cleaving ribozyme (sTRSV) and a hairpin structure to expose the ribosome binding site. It can remove unnecessary RNA sequences from different transcription start sites, providing insulation for synthetic circuits from genetic context. |
75 bp |
| BBa_K3395008 | PcaUAM | In the upstream generator use in the PCA control system, the function of this element is to generate PcaUAM protein, which is can activate the downstream promoter: p3b5b | 837 bp |
| BBa_K3395009 | pPCA_3B5B | The promoter itself is not stable in binding to the RNA polymerase , but when the PcaUAM protein is present, its binding to the RNA polymerase will be easier. | 93 bp |
| BBa_B0030 | hTPH1 RBS | The ribosome binding site (RBS) sequence serves as a rendezvous point for the ribosome, which moves along the mRNA and proceeds to translate mRNA from the following start codon. | 21 bp |
| BBa_B0029 | hPCBD1 RBS | The ribosome binding site (RBS) sequence serves as a rendezvous point for the ribosome, which moves along the mRNA and proceeds to translate mRNA from the following start codon. | 20 bp |
| BBa_B0035 | hQDPR RBS | The ribosome binding site (RBS) sequence serves as a rendezvous point for the ribosome, which moves along the mRNA and proceeds to translate mRNA from the following start codon. | 20 bp |
| BBa_K5040000 | Zif268-rlinker-m-hTPH1 | The fusion protein of m-hTPH1 and Zif268 binding domain with 36 bp rigid linker. m-hTPH1 is the monomeric form of the hTPH1 gene, harboring a deletion of the first 99 N-terminal and last 24 C-terminal amino acids, NΔ99/CΔ24. Zif268 recognizes the 9 bp sequence: 5'-GCG TGG GCG-3'. |
1296 bp |
| BBa_K5040001 | PBSII-rlinker-hPCBD1 | The fusion protein of hPCBD1 and PBSII binding domain with 36 bp rigid linker. hPCBD1 dehydrates the BH3OH to the qBH2, acting as an enzyme in the BH4 regeneration system. PBSII recognizes the 9 bp sequences: 5'-GTG TGG AAA-3' |
694 bp |
| BBa_K5040002 | ZFa-hQDPR | The fusion protein of hQDPR and ZFa binding domain. hQDPR reduces the qBH2 to the BH4, acting as an enzyme in the BH4 regeneration system. ZFa binds the 9 bp sequence: 5'-GTC GAT GCC-3' |
1072 bp |
| BBa_K5040003 | T7hyb1 terminator | The T7 terminator is a sequence from bacteriophage T7, allowing efficient transcription termination. The T7 promoter and terminator are commonly used to regulate gene expression of recombinant proteins. A modified T7 terminator sequence with a UUCG loop and G-C base pair substitutions improved termination efficiency by 40% in vitro. |
42 bp |
| Part number | Name | Description | Length |
|---|---|---|---|
| BBa_K5040004 | RCORI105-ZNF motifs-RCORI65 | The version is composed of RCORI-65, ZNF motifs, and RCORI-105. | 365 bp |
| BBa_K5040005 | pLacIQ-PcaUAM-T7Hyb1 terminator | The expression cassette is composed of pLacIQ, RBS, PcaUAM, T7Hyb terminator | 964 bp |
| BBa_K5040006 | pPCA3B5B-RiboJ-mGL | The expression cassette is composed of pPCA3B5B, RiboJ, mGL, and T7 terminator. | 922 bp |
| BBa_K5040007 | pPCA3B5B-RiboJ-RepA | The expression cassette is composed of pPCA3B5B, RiboJ, RepA, and T7 terminator. | 889 bp |
| BBa_K5040008 | T7promoter-Zif268-rLink-m-hTPH1-PBSII-rlinker-hPCBD1-ZFa-hQDPR-T7terminator | The expression cassette is composed of T7promoter, Zif268-rLink-m-hTPH1, PBSII-rlinker-hPCBD1, ZFa-hQDPR, T7terminator | 3203 bp |