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Synthetic biology provides a diverse set of orthogonal elements for assembling complex biological systems, which require modular design and systematic characterization for efficient construction. Our project aims to develop an intestinal probiotic-based system capable of sensing environmental signals and producing the highly stable therapeutic peptide GLP-1. To achieve this, we have integrated a wide range of sensing components and protein engineering systems, enabling precise spatiotemporal regulation of bacterial viability and metabolic activity, both actively and passively. To this end, we have designed a series of basic and composite parts, along with specialized plasmids to support accurate control and functional implementation within the biological system.
The basic parts developed in this project include promoters and coding elements that form the core framework of our system. Among the promoters used are the protocatechuic acid (PCA)-inducible promoter PLV (BBa_25Y8JP8F), the glucose uptake negative-responsive promoter GURB3-2 (BBa_25TRS3OS), and the temperature-sensitive promoter PtlpA (BBa_25RFWS4Y ). Coding elements comprise the PCA-responsive transcription repressor PcaV (BBa_25OBUNJP), orthogonal split SspGyrB-ECF16 fusion protein halves, and the sequence-optimized temperature-responsive repressor TlpA* (BBa_25XF4HZ5). Additionally, the protein engineering module includes orthogonal aminoacyl-tRNA synthetases and their cognate tRNAs, as well as a superfolder GFP (sfGFP) reporter protein containing amber codons (BBa_25R2L2YD). Together, these basic components establish the foundational framework of the project.
Composite parts integrate multiple basic components to achieve complex regulatory logic and sensing functions. For instance, the PCA-responsive operon (BBa_25N5FWAK) enables sensing and induction in response to PCA, a metabolite derived from green tea. An upstream AND-gate (BBa_2581ND93), constructed based on orthogonal split intein trans-splicing, allows spatiotemporal control in response to multiple input signals. The temperature-responsive self-repressive system (BBa_2575N2V6) and the tandem dual-promoter element incorporating the insulator RiboJ (BBa_2552PNC9) provide additional flexibility for implementing OR-gate logic. Furthermore, orthogonal aaRS/tRNA pairs (BBa_2565RBMY and BBa_2543FT42) enable site-specific incorporation of non-canonical amino acids in E. coli via genetic code expansion, broadening the application of ucAAs in custom functional protein design for synthetic biology.
For plasmid construction, we utilized the pUltra backbone (BBa_25A3A634), a high-efficiency vector for site-specific incorporation of non-canonical amino acids in E. coli. This system provides the essential genetic framework for introducing orthogonal aaRS/tRNA pairs and optimizing ncAA incorporation efficiency.
To enable robust and tunable control over bacterial viability, we designed a dual-input OR-gate genetic circuit responsive to temperature and arabinose. This composite part integrates a temperature-sensitive self-repressive system with a tandem dual-promoter construct, achieving an initial ON/OFF expression ratio of approximately 5-fold. The system can be further coupled with promoter optimization and toxin-antitoxin modules to implement programmable bacterial suicide.
This composite part provides a modular framework and practical reference for teams developing OR-gate systems. It demonstrates how complex operons can be orthogonally rewired through optimized regulatory components, and serves as a foundation for constructing more sophisticated circuits by integration with AND-gates or amplifier modules.
For more details, please visit our Registry Page （BBa_25NKC5AV） and Engineering Page in Part 3 iterations.
| Part Number | Name | Type | Brief Introductions |
|---|---|---|---|
| BBa_25Y8JP8F | PLV | Promoter | A protocatechuic acid (PCA)-inducible promoter PLV. |
| BBa_25OBUNJP | PcaV | Coding | The protocatechuic acid (PCA)-responsive transcriptional repressor PcaV. |
| BBa_25YZWH1A | SspGyrBC-ECF16C | Coding | A fusion protein comprising the C-terminal halves of split intein SspGyrB and split ECF16. |
| BBa_259H9ZPU | SspGyrBN-ECF16N | Coding | A fusion protein comprising the N-terminal halves of split intein SspGyrB and split ECF16. |
| BBa_25RT9PC8 | P16 | Promoter | The ECF16-cognate promoter P16. |
| BBa_25TRS3OS | GURB3-2 promoter | Promoter | A glucose uptake negative-response promoter GURB3-2. |
| BBa_25XF4HZ5 | TlpA* | Coding | The temprature-responsive transcriptional repressor TlpA*. |
| BBa_25RFWS4Y | PtlpA with RBS | Promoter | The temprature-responsive promoter PtlpA with its cognate RBS. |
| BBa_25NOQJ8F | PBAD promoter | Promoter | Minimal arabinose-inducible promoter PBAD. |
| BBa_25XUNXCE | RiboJ | Coding | Self-cleaving ribozyme RiboJ as an insulator in bacterial systems. |
| BBa_25FJQXDY | S-GLP-1Z-CPP | Coding | A fusion peptide with a signal peptide, GLP-1Z, a cell-penetrating peptide and a C-terminal His-tag. |
| BBa_2504KELH | SUMO-GLP-1Z-CPP | Coding | A fusion peptide with N-terminal His-tag, SUMO-tag, GLP-1Z and a cell-penetrating peptide(CPP). |
| BBa_25DRMK5X | SUMO-GLP-1 | Coding | A fusion peptide with N-terminal His-tag, SUMO-tag, and codon-optimized human GLP-1 (7-36) sequence. |
| BBa_25QIIBEZ | 5-HTP aaRS | Coding | Orthogonal 5-hydroxytryptophan (5-HTP) aminoacyl-tRNA synthetase. |
| BBa_25LSICU8 | MjtRNAsTyr | Coding | Orthogonal tRNA for transferring sulfotyrosine (sTyr). |
| BBa_255G11B4 | SctRNA5-HTP | Coding | Orthogonal tRNA for transferring 5-hydroxytryptophan (5-HTP). |
| BBa_259WRPM0 | sTyr aaRS | Coding | Orthogonal sulfotyrosine (sTyr) aminoacyl-tRNA synthetase. |
| BBa_25R2L2YD | sfGFP reporter protein harboring amber codons | Coding | sfGFP reporter protein harboring two amber codons. |
| BBa_252MVYLF | proK promoter | Promoter | The proK promoter is a natural strong promoter in E. coli, primarily driving efficient tRNA gene transcription and used for tRNA expression cassettes. |
| Part Number | Name | Type | Brief Introductions |
|---|---|---|---|
| BBa_25N5FWAK | J23105-PcaV-PLV | Device | A protocatechuic acid (PCA)-responsive operon. |
| BBa_2581ND93 | J23105-PcaV-PLV-SspGyrBC-ECF16C-J23101-RamA-PacrRA-SspGyrBN-ECF16N | Device | An upstream AND-gate circuit based on split intein trans-splicing with SspGyrB-ECF16. |
| BBa_25MR74ON | P16-GFP | Reporter | A GFP reporting circuit for ECF16 expression. |
| BBa_2575N2V6 | PtlpA-tlpA* | Device | The temperature-responsive ptlpA-tlpA*self-repressive system. |
| BBa_2552PNC9 | J23101-PBAD-RiboJ | Promoter | Tandem dual-promoter element with the insulator RiboJ |
| BBa_25NKC5AV | PtlpA-tlpA*-araC-J23101-PBAD-RiboJ | Device | A dual-input OR-gate circuit responsive to arabinose and temperature |
| BBa_2565RBMY | Orthogonal 5-HTP aaRS and tRNA | Coding | 5-HTP aaRS can pair with SctRNA5-HTP to form orthogonal aminoacyl-tRNA synthetase/tRNA pairs. |
| BBa_2543FT42 | Orthogonal sTyr aaRS and tRNA | Coding | sTyr aaRS can pair with MjtRNAsTyr to form orthogonal aminoacyl-tRNA synthetase/tRNA pairs. |
| Part Number | Name | Type | Brief Introductions |
|---|---|---|---|
| BBa_25A3A634 | pUltra | Plasmid | The pUltra plasmid is a high-efficiency tool for site-specific incorporation of non-canonical amino acids in E. coli. |