Parts
Introduction
CAU-China has registered 34 basic parts and 16 composite parts during this year’s wet lab work.
These components form three primary functional circuits, responsible for controlled toxin delivery, the Dual Safety Strategy as well as Reprogramming of Tail fiber.
Innovation
By utilizing the inherent DNA packaging mechanism of the T7 bacteriophage’s head capsid, we packaged the toxin plasmid we constructed (instead of the native genome) to synthesize phage-like particles (PLPs), while eliminating the self-replication and bacterial lysis properties of natural bacteriophages. We avoided the risk of uncontrollable proliferation associated with natural bacteriophages and leveraged the advantage of their specific targeting specificity, ensuring that PLPs are unable to produce progeny PLPs and yet possess targeted bactericidal activity.
Furthermore, we introduced a phage tail fiber replacement strategy to address the limitation of the narrow host range of wild-type (WT) T7 bacteriophages. This modification enables PLPs to acquire the capability of specifically targeting other pathogenic bacteria of interest. Overall, this design broadens the range of target recognition for PLPs and enhances the flexibility of their applications.
In controlled toxin delivery, we employed the MazF-MazE toxin-antitoxin system. MazF is a stable ACA sequence-specific mRNA endoribonuclease that irreversibly cleaves intracellular mRNA, thereby causing the termination of protein synthesis, disruption of metabolic activities, and ultimately leading to cell death. This design facilitates the broad-spectrum killing of pathogenic bacteria, which is unconstrained by the structural characteristics of pathogenic bacteria, and lays the foundation for the subsequent replacement of the tail fiber module to kill other target bacterial strains.
In the process of packaging the toxin plasmid, we employed the packaging sequence of the T7 bacteriophage. This design ensures that the packaging process is regulated by T7 RNA polymerase. More critically, the packaging sequence is specifically recognized and then cleaved by terminase—this facilitates the accurate cleavage of large concatemers amplified via rolling circle replication, and further promotes the specific packaging of the target sequence by the head capsid.
In the Dual Safety Strategy, a pKD46-SacB recombinant system was designed, and the method for inserting the target fragment into chassis bacteria via double-crossover homologous recombination was optimized. Utilization of double screening facilitates more accurate acquisition of chassis bacteria with successful insertion of the target fragment.
In terms of synthesizing phage-like particle capsids, we only removed the genes associated with self-replication and bacterial lysis. After separately isolating the gp1 gene (which encodes T7 RNA polymerase) from the packaging sequence, the remaining genomic fragments were divided into 4 major categories and sequentially inserted into the chassis bacterial genome. This design helps ensure the integrity and controllability of the phage-like particles, making the bactericidal application of phage-like particles more stable and safer.
In terms of inducing the initiation of phage-like particles synthesis, an IPTG-inducible promoter (responsive to the exogenously added inducer IPTG) was inserted upstream of the gp1 gene. This design facilitates the control of PLP synthesis, enhances safety, and reduces the early-stage growth burden of chassis bacteria.
In Reprogramming of Tail fiber, the native T7 tail fiber gene was replaced with a phage tail fiber gene capable of recognizing non-T7 host bacteria. This design facilitates the verification of the replaceability and recognition specificity of tail fibers, and further enables flexible and broad-spectrum modification and application.
Basic Parts
| Part name | Registry Code | Part Type | Brief introductions |
|---|---|---|---|
| tetR/tetA promoters | BBa_K4028005 | promoter | Promoter responsive to tetR signals, inactivates downstream gene transcription under tetR conditions and becomes active in the absence of tetR. |
| MazE | BBa_25M9TY8W | Coding | The cognate antitoxin of the MazEF bacterial toxin-antitoxin system. |
| MazF | BBa_K3964004 | Coding | Toxin gene in the toxin-antitoxin system. Encodes a stable ACA sequence-specific mRNA endoribonuclease. |
| ΦOR | BBa_259URUSG | promoter | The origin of plasmid rolling-circle replication, and a promoter that initiates rightward transcription. |
| pacB | BBa_251FWZTL | Regulatory | The specific recognition site for DNA packaging by the protein capsid. |
| pacC | BBa_25PRX72M | Signalling | A specific sequence recognized by terminases, which enables the cleavage of large concatemers. |
| DTR | BBa_25G4SN4M | Scar | Contributes to the maintenance of genomic integrity and stability. |
| tetR | BBa_25N4X1W9 | Coding | Transcriptional Repressor of Tetracycline Inducible Promoter tetR/tetA promoters. |
| J23119 | BBa_K5303058 | promoter | Constitutive promoter for maintaining downstream gene transcription. |
| RBS1 | BBa_K3143683 | RBS | Ribosome binding site, ensures proper expression of MazE gene. |
| RBS2 | BBa_25ZZT88Y | RBS | Ribosome binding site, ensures proper expression of MazF gene or bjGFP gene. |
| RBS3 | BBa_K3793001 | RBS | Ribosome binding site, ensures proper expression of tetR gene. |
| RBS4 | BBa_25BZM5SS | RBS | Ribosome binding site, ensures proper expression of downstream gene. |
| pBAD | BBa_K4657003 | promoter | Promoter of the L-arabinose operon of E. coli; the araC regulatory gene is transcribed in the opposite direction. |
| araC | BBa_25BIPQ9T | Coding | Encodes L-arabinose regulatory protein. |
| gp18 | BBa_25FFFT6N | Coding | Encodes the TerS protein, which serves as the small subunit of terminase and forms the holoterminase complex together with TerL. |
| gp19 | BBa_258O20J7 | Coding | Encodes the TerL protein, which serves as the large subunit of terminase and forms the holoterminase complex together with TerS. |
| T7 Terminator | BBa_2556TVGQ | Terminator [SO:0000141] | Terminator ensures proper termination of gene expression. |
| MG1655_up arm terR-MazE | BBa_253I5JX5 | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the chassis genome. |
| MG1655_down arm terR-MazE | BBa_25I7RSFG | Homologous Region [SO:0000853] | Downstream homology arm for integrating the sequence into the chassis genome. |
| pLac | BBa_25JOVGFL | promoter | Promoter responsive to IPTG signals, activates downstream gene transcription under IPTG conditions and becomes inactive in the absence of IPTG. |
| gp1 | BBa_252JG2ZB | Coding | Encodes T7 RNA polymerase. |
| bjGFP | BBa_K3796205 | Coding | Encodes reporter fluorescent protein. |
| SacB | BBa_25H4Z185 | Coding | Encodes levansucrase, as a negative selection marker in dual-selection experiments. |
| SacB promoter | BBa_K4818055 | promoter | Constitutive promoter for maintaining SacB gene transcription. |
| MG1655_up arm gp1 | BBa_25YKS3F2 | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the chassis genome. |
| MG1655_down arm gp1 | BBa_259G0YEY | Homologous Region [SO:0000853] | Downstream homology arm for integrating the sequence into the chassis genome. |
| T7g-1-1 | BBa_25N06Z7C | T7 | DNA fragment representing the portion preceding the removal of gp1 from the T7 phage Class I genes. |
| T7g-1-2 | BBa_25Y3R1A1 | T7 | DNA fragment derived from the T7 phage Class I genes after removing gp1. |
| T7g-2 | BBa_25N8F0B4 | T7 | The gene segment of the T7 phage genome associated with replication—the Class II genes—encoding 26 proteins. |
| T7g-3 | BBa_25UMTMP5 | T7 | The region of the phage genome associated with structural genes—the first part of the Class III genes, comprising 11 proteins (gp6.5-gp12). |
| T7g-4-1 | BBa_25TF4YQ1 | T7 | The middle portion of the phage genome’s structural gene region—the Class III genes—comprising 12 proteins (gp13-gp19). |
| T7g-4-2 | BBa_25QXUND3 | T7 | The final segment of the phage genome’s structure-related region—the Class III genes—remaining after removal of pacB, pacC, and DTR, encoding the gp19.5 protein. |
| MG1655_up arm T7 | BBa_25LAGW4U | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the chassis genome. |
| MG1655_down arm T7 | BBa_256RELF9 | Homologous Region [SO:0000853] | Downstream homology arm for integrating the sequence into the chassis genome. |
| T7-Up arm-1 | BBa_25Y23WPG | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the chassis genome. |
| T7-Up arm-2 | BBa_25NJSPXC | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the chassis genome. |
| T7-Up arm-3 | BBa_25EVE7SP | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the chassis genome. |
| T7 gp17 up arm | BBa_25MY0E0A | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the T7 genome. |
| T7 gp17(149) up arm | BBa_250LB4N5 | Homologous Region [SO:0000853] | Upstream homology arm for integrating the sequence into the T7 genome. |
| phi psa 17 VO98_215 | BBa_256RYFBB | Coding | Encodes the intact tail-fiber protein of Pseudomonas syringae phage ΦPSA17. |
| phi psa 17 VO98_215(149-) | BBa_25DLXM3E | Coding | Encodes the C-terminal receptor-binding half of the Phi PSA17 tail-fiber protein. |
| T7 gp17 down arm | BBa_25NGZJMZ | Homologous Region [SO:0000853] | Downstream homology arm for integrating the sequence into the T7 genome. |
| 6x Histidine tag | BBa_25PJFRK8 | Coding | Encodes a 6x Histidine tag for protein purification and isolation using Ni-NTA purification. |
Composite Parts
| Composite Part Name | Registry Code | Part Type |
|---|---|---|
| araC-pBAD-Ter | BBa_25DXPDE7 | Regulatory |
| araC-pBAD-MazE-Ter | BBa_25EFERKZ | Regulatory |
| araC-pBAD-MazF-Ter | BBa_251RZ4UA | Regulatory |
| araC-pBAD-MazE-MazF-Ter | BBa_25M3U0AD | Regulatory |
| ΦOR-pacB-DTR-pacC-pTetA-MazF-Ter | BBa_25CP9YBT | Regulatory |
| ΦOR-pacB-DTR-pacC-pTetA-bjGFP-Ter | BBa_25W32O4M | Regulatory |
| J23119-TetR-MazE-Ter | BBa_25PC6LXU | Regulatory |
| pLac-gp1-bjGFP-Ter | BBa_25YN2442 | Regulatory |
| MG1655_up arm T7_T7g-1-1_T7g-1-2_MG1655_down arm T7 | BBa_25DQKZPR | Homologous Region [SO:0000853] |
| T7-Up arm1_T7g-2_MG1655_down arm T7 | BBa_255T8O5T | Homologous Region [SO:0000854] |
| T7-Up arm2_T7g-3_MG1655_down arm T7 | BBa_25GTPRZQ | Homologous Region [SO:0000855] |
| T7-Up arm3_T7g-4-1_T7g-4-2_T7 Terminator_MG1655_down arm T7 | BBa_25U7A4NS | Homologous Region [SO:0000856] |
| gp17 up arm-VO98_215-6xHis-gp17 down arm | BBa_251JH9IC | Homologous Region [SO:0000857] |
| gp17 up arm-6xHis-VO98_215-gp17 down arm | BBa_25P8JRVM | Homologous Region [SO:0000858] |
| gp17（149） up arm-VO98_215（149-）-6xHis -gp17 down arm | BBa_25BGJ4CZ | Homologous Region [SO:0000859] |
| gp17（149） up arm-6xHis-VO98_215（149-）-gp17 down arm | BBa_256K6W59 | Homologous Region [SO:0000860] |
Cloning Vectors
| Part Name | Registry Code | Origin | Length (bp) |
|---|---|---|---|
| pUC19 | BBa_K4354011 | Artificial synthesized from Joachim Messing, 1983 | 2686 |
| pKD46 | BBa_K5313034 | Artificial synthesized from Datsenko and Wanner, 2000 | 6329 |
Part Collections
| Name | UUID |
|---|---|
| packaging | 5e3aa590-c37e-4923-a9f3-a57d0023bef7 |
| Reprogramming of Tail fiber | 85d0a553-6eee-4b82-84d0-b24733928321 |