Overview
The V-CHARGEs platform establishes a modular system for efficient ATP regeneration in cell-free biosynthesis. By leveraging Virus-Like Particle (VLP) encapsulation, V-CHARGEs stabilizes the high-activity polyphosphate kinase SlPPK while simultaneously coupling ATP-consuming enzymes on the VLP exterior. This design transforms V-CHARGEs into a "nanocharging station" that continuously fuels diverse biocatalytic reactions with low-cost polyphosphate as the sole energy input.
Specifically, the system integrates three synergistic components:
(1) SlPPK-SP fusion proteins that anchored inside P22-VLPs, where the capsid protects its enzymatic activity and stability;
(2) CP-SpyTag fusion proteins that assemble into the VLP and display reactive SpyTag on the exterior.
(3) SpyCatcher-enzyme fusions that enable modular attachment of diverse ATP-consuming enzymes to the particle surface.
Through this architecture, ADP and polyphosphate diffuse freely into the VLP, where SlPPK catalyzes ATP regeneration. The resulting ATP rapidly diffuses outward and is directly consumed by surface-anchored enzymes, establishing a continuous ATP/ADP cycle with polyphosphate replenishment.
To overcome long-standing industrial bottlenecks, V-CHARGEs provides protection against phosphate inhibition, pH fluctuations, thermal instability, and proteolysis-limitations that have hindered previous PPK-based ATP regeneration systems. Beyond stabilization, the modular design enables flexible coupling of multiple enzymes, creating artificial multi-enzyme complexes with enhanced efficiency through substrate channeling.
The V-CHARGEs platform was developed in an iterative DBTL(Design-Build-Test-Learn) cycle that integrated structural design, in vitro characterization, and functional validation in cell-free production pathways. We demonstrated its versatility by powering reactions ranging from firefly luciferase-driven bioluminescence to nucleotide precursor synthesis (e.g., 5'-CMP) and antioxidant biosynthesis (e.g., glutathione). Together, these results highlight V-CHARGEs as a robust, scalable, and generalizable platform for cell-free biomanufacturing, offering a new route to industrial energy supply in synthetic biology.
The part collection of V-CHARGEs consists of three categories:
1. Basic Parts: The biobricks of the whole project. The cornerstones for us to achieve efficient ATP regeneration.
2. Composite Parts: Our newly designed biological components. The backbone of our project.
3. Primers: Important tools we used in our DNA lab. Playing an indispensable role in experiments.
1. Basic Parts
Table 1. Basic Parts of Our V-CHARGEs.
| Part Number | Name | Description |
|---|---|---|
| BBa_25HTMYNU | Sulfurovum lithotrophicum Polyphosphate Kinase (SlPPK) | An enzyme that can convert ADP to ATP using polyphosphate. |
| BBa_25HRNXA3 | Bacteriophage P22 Scaffold Proteins (142-303) | A portion of the structure of Bacteriophage P22 Scaffold Proteins can bind to the Bacteriophage P22 Coat Protein, enabling the encapsulation of the fused protein within the VLP particles formed by the Bacteriophage P22 Coat Protein. |
| BBa_252A0TI3 | Bacteriophage P22 Coat Protein(CP) | A protein particle capable of self-assembling into a Virus-Like Particle (VLP) with a diameter of approximately 50 nm, which can interact with Bacteriophage P22 Scaffold Proteins(SP), encapsulating the fusion protein linked to the Scaffold Protein inside the VLP protein particle. |
| BBa_2561SO8G | Firefly Luciferase(FLuc) | Firefly luciferase is an enzyme that catalyzes the oxidation of luciferin to produce bioluminescence. |
| BBa_25KENB1A | Uridine-Cytidine Kinase(UCK) | Uridine-Cytidine Kinase (UCK) is an enzyme that phosphorylates cytidine to produce 5'-CMP, a critical step in pyrimidine nucleotide metabolism for nucleic acid synthesis. |
| BBa_25LWEJG7 | γ-Glutamylcysteine Synthetase(γ-GCS) | γ-Glutamylcysteine Synthetase(γ-GCS) is an enzyme that catalyzes the formation of γ-glutamylcysteine from glutamate and cysteine, a key step in glutathione(GSH) biosynthesis. |
| BBa_25R73ZP5 | Glutathione Synthetase(GS) | Glutathione Synthetase(GS) is an enzyme that catalyzes the final step in glutathione biosynthesis, combining γ-glutamylcysteine with glycine to produce glutathione(GSH). |
| BBa_25DKD8NM | SpyTag | SpyTag is a peptide that covalently binds SpyCatcher, enabling specific protein conjugation for applications like vaccine development and protein engineering. |
| BBa_25NVAFAN | SpyCatcher | SpyCatcher is a peptide that covalently binds SpyTag, enabling specific protein conjugation for applications like vaccine development and protein engineering. |
| BBa_25XJKPW5 | SGGG linker2 | GS linker is a flexible peptide sequence, typically composed of glycine and serine residues, used to connect protein domains while maintaining their independent function and structural stability. |
| BBa_K1362443 | His-tag | His-tag is a short peptide tag widely used in recombinant protein expression and purification, typically consisting of six consecutive histidine residues. |
| BBa_K4587111 | FLAG tag | The peptide tag FLAG has high specificity for its commercial antibodies, which simplifies the target protein detection. |
| BBa_K5487118 | TEV site | Tobacco Etch Virus (TEV) protease recognition and cleavage site. |
| BBa_K3457003 | T7 promotor | T7 promoter of pET-modification vector. |
| BBa_K4657003 | araBAD promotor | araBAD promoter is an E. coli promoter that is tightly controlled by inducer L-arabinose and repressor Arac. |
| BBa_K1222999 | lac operator | The lacO operator is a DNA sequence that is recognized by the LacI repressor. |
| BBa_K3189003 | T7 terminator | A transcription terminator for bacteriophage T7 RNA polymerase. |
| BBa_K3033016 | rrnB T1 terminator | Derived from pBAD24, and synthesized through IDT. This codes for a rrnB T1 Terminator site. |
| BBa_K4634001 | RBS | Efficient Ribosome Binding Site (RBS) from bacteriophage T7 gene 10. |
2. Composite Parts
Table 2. Composite Parts of Our V-CHARGEs.
| Part Number | Name | Description |
|---|---|---|
| BBa_25R8SDG4 | SIPPK-SP-FLAG-TEV-His expression system(T7) | This pathway expresses the SlPPK-SP-FLAG-TEV-His fusion protein under lPTG-induced conditions. |
| BBa_25SAVPX0 | SlPPK-SP-FLAG-TEV-His expression system(pBAD) | This pathway expresses the SIPPK-SP-FLAG-TEV-His fusion protein under Arabinose-induced conditions. |
| BBa_25GLM18X | SlPPK-SP-FLAG expression system(pBAD) | This pathway expresses the SIPPK-SP-FLAG fusion protein under Arabinose-induced conditions. |
| BBa_250ADUTI | CP-His-SpyTag expression system(T7) | This pathway expresses the CP-His-SpyTag fusion protein under IPTG-induced conditions. |
| BBa_25BNN8IN | CP-SpyTag expression system(T7-pACYC) | This pathway expresses the CP-SpyTag fusion protein under IPTG-induced conditions. |
| BBa_25QTUT0S | SlPPK-SP-FLAG, CP-SpyTag co-expression system(T7-pACYC) | This pathway expresses the SlPPK-SP-FLAG and CP-SpyTag fusion protein under IPTG-induced conditions. |
| BBa_25N4XQ2O | FLuc-SpyCatcher expression system(T7) | This pathway expresses the FLuc-SpyCatcher fusion protein under IPTG-induced conditions. |
| BBa_252IOOB5 | UCK-SpyCatcher expresssion system(T7) | This pathway expresses the UCK-SpyCatcher fusion protein under IPTG-induced conditions. |
| BBa_25LO0B5F | γ-GCS-SpyCatcher expression system(T7) | This pathway expresses the γ-GCS-SpyCatcher fusion protein under IPTG-induced conditions. |
| BBa_25SOUJ6Y | GS-SpyCatcher expression system(T7) | This pathway expresses the GS-SpyCatcher fusion protein under IPTG-induced conditions. |
3. Primers
Table 3. Primers We Have Used.
| Part Number | Name | Description |
|---|---|---|
| BBa_251XAR6T | SlPPK-SP-FLAG(-TEV-His)_pBAD_F | These primer is designed to amplify SlPPK-SP-FLAG and SlPPK-SP-FLAG-TEV-His fragments, enabling its insertion into the pBAD33 plasmid digested with HindIII. |
| BBa_25WRNDBF | SlPPK-SP-FLAG _pBAD_R | |
| BBa_25SGDDH1 | SlPPK-SP-FLAG -TEV-His_pBAD_R | |
| BBa_251AYLGK | SIPPK-SP_F | This primer is used to verify whether SlPPK-SP fragment has been successfully constructed. |
| BBa_25QHBXZZ | SIPPK-SP_R | |
| BBa_25JPCTF4 | γ-GCS_F | This primer is used to verify whether UCK-SpyCatcher,γ-GCS-SpyCatcher，GS-SpyCatcher DNA fragment has been successfully constructed. |
| BBa_25XNQABZ | GS_F | |
| BBa_25S8NNR6 | UCK_F | |
| BBa_25O5MPMF | SpyCatcher_R | |
| BBa_25WCQGP5 | SlPPK-SP-His_pBAD_F | This primer is designed to amplify SlPPK-SP-His DNA fragment, enabling its insertion into the pBAD33 plasmid digested with HindIII. |
| BBa_25BPAPI3 | SlPPK-SP-His_pBAD_R | |
| BBa_254LF5QL | CP-SpyTag-His_pBAD_F | This primer is designed to amplify CP-SpyTag-His DNA fragment, enabling its insertion into the pBAD33 plasmid digested with SalI. |
| BBa_25NXERRO | CP-SpyTag-His_pBAD_R | |
| BBa_25KFB198 | CP-SpyTag-His_pACYC_F | This primer is designed to amplify CP-SpyTag-His DNA fragment, enabling its insertion into the pACYCDuet-1 plasmid digested with SalI. |
| BBa_25VFE4GL | CP-SpyTag-His_pACYC_R | |
| BBa_25FVUBBD | SlPPK-SP_pACYC_F | This primer is designed to amplify SlPPK-SP DNA fragment, enabling its insertion into the CP_pACYCDuet-1 plasmid digested with BglII and XhoI. |
| BBa_25KRUHJO | SlPPK-SP_pACYC_R | |
| BBa_25H9AZCL | UCK_pET28a_F | This primer is designed to amplify UCK DNA fragment, enabling its insertion into the pET28a plasmid digested with NcoI and HindIII. |
| BBa_25HUZDAD | UCK_pET28a_R | |
| BBa_2598UWXW | γ-GCS_pET28a_F | This primer is designed to amplify γ-GCS DNA fragment, enabling its insertion into the pET28a plasmid digested with NcoI and HindIII. |
| BBa_25BFDFUD | γ-GCS_pET28a_R | |
| BBa_254ZNN6J | GS_pET28a_F | This primer is designed to amplify GS DNA fragment, enabling its insertion into the pET28a plasmid digested with NcoI and HindIII. |
| BBa_253H0ZRG | GS_pET28a_R |