Parts
Our comprehensive composite of biological parts and devices that enable orthogonal DNA replication, SpyTag-SpyCatcher interactions, and modular protein assembly systems in E. coli.
Experiment A
This part builds an orthogonal DNA replication system in E. coli that can replicate and evolve linear DNA replicons independently from the host genome.
The design includes two connected modules:
pET-IDT_Operon plasmid (BBa_25EGZQUG) – provides the replication machinery. It contains two inducible operons. The first operon (Ptac-IPTG) expresses the wild-type O-DNAP (P1) (BBa_25E979WJ) together with TP (P13) (BBa_257P3IH0) and SSB (P12) (BBa_25IIG0HB) to start normal orthogonal replication. The second operon (PrhaBAD) expresses a mutant O-DNAP (N71D) (BBa_25U84DWA), which has reduced proofreading activity and can introduce mutations specifically into the linear replicon. This control system lets us switch between accurate replication and mutagenic replication by adding different inducers.
Linear O-Replicons (sfGFP or SpyCatcher) – serve as orthogonal templates and readouts. Each linear replicon is flanked by PRD1 inverted terminal repeats (ITRs) (BBa_25QMSXHV) for TP-initiated replication and includes a constitutive promoter for gene expression. The sfGFP version reports replication efficiency through fluorescence, while the SpyCatcher version tests whether the system can maintain and express a functional protein.
Overall, this system allows independent replication, targeted mutagenesis, and functional screening of linear DNA inside E. coli.
Figure 1. Experiment A parts design schematic diagram.
Created with BioRender.
| Item | Number | Name | Type | Description |
|---|---|---|---|---|
| 1 | BBa_253IFO11 | spycatcher | Protein coding | Covalent binder for SpyTag fusion |
| 2 | BBa_25QMSXHV | ITRs(right) | DNA element | PRD1 inverted terminal repeat origin |
| 3 | BBa_25C8A8UB | AmpR | Selectable marker | Ampicillin resistance gene |
| 4 | BBa_25YI441T | sfGFP | Reporter gene | Superfolder GFP for fluorescence |
| 5 | BBa_255SJV1X | pET-IDT | Plasmid backbone | T7-based expression vector |
| 6 | BBa_25IVX5PZ | Ptac-IPTG promoter | Promoter | IPTG-inducible hybrid Ptac promoter |
| 7 | BBa_257P3IH0 | TP (P13) | Protein coding | Terminal protein initiating replication |
| 8 | BBa_25E979WJ | O-DNAP(P1) | Protein coding | wild type DNA polymerase for replicon |
| 9 | BBa_25U84DWA | O-DNAP(N71D) | Protein coding | mutant DNA polymerase for replicon |
| 10 | BBa_25IIG0HB | SSB (P12) | Protein coding | Single-stranded DNA binding protein |
| 11 | BBa_25O5LVE3 | PrhaBAD promoter | Promoter | Rhamnose-inducible promoter for gene control |
| 12 | BBa_25MAH6KB | terminator | DNA element | Transcriptional stop signal |
| 13 | BBa_254YLUTH | o-replicon-sfGFP | Linear DNA | Orthogonal replicon carrying sfGFP |
| 14 | BBa_25C90YHE | O-replicon-SpyCatcher | Linear DNA | Orthogonal replicon carrying SpyCatcher |
| 15 | BBa_25EGZQUG | pET-IDT_Operon | Plasmid construct | Synthetic operon with TP, O-DNAP, SSB |
Experiment B
This experiment is designed to create a SpyTag–SpyCatcher interaction platform based on the split luciferase complementation assay (SLCA).
Each construct is built on the pET-IDT backbone (BBa_255SJV1X) and combines N-terminal (BBa_25W10VKK) or C-terminal (BBa_25CVCYGH) fragments of Renilla Green luciferase (RenG) with SpyTag or SpyCatcher, allowing us to detect covalent binding events through luminescence recovery.
Three devices were constructed:
SpyTag factories (BBa_25NAGCBQ) – expressing either N′- or C′-terminal halves of RenG fused to SpyTag (BBa_25PTH3PS).
SpyCatcher factories – expressing SpyCatcher or its engineered mutants (BBa_257J1PEJ, BBa_25E2PO9G, BBa_25F2XK6E, BBa_25CJBY99, BBa_25NYARGM, BBa_25AF6R01) fused to the complementary RenG fragment.
SpyCatcher factory-no luciferase (BBa_250WYFV2) – expressing only SpyCatcher with a His-tag as a baseline control.
When a SpyTag-RenG fragment and a SpyCatcher-RenG fragment are co-expressed, covalent SpyTag–SpyCatcher binding brings the two RenG halves into proximity, reconstituting an active luciferase enzyme that emits light. The luminescence intensity therefore reflects the binding efficiency and kinetics between different SpyCatcher variants and SpyTag.
By linking split luciferase reporting with SpyTag–SpyCatcher binding, this system provides a simple and sensitive method to evaluate how mutations affect interaction efficiency. It also enables quick and quantitative comparison of different SpyCatcher mutants under the same conditions.
Figure 2. Experiment B parts design schematic diagram.
Created with BioRender.
| Item | Number | Name | Type | Description |
|---|---|---|---|---|
| 1 | BBa_255SJV1X | pET-IDT | Plasmid backbone | T7-based expression vector |
| 2 | BBa_25W10VKK | N'-RenG luciferase | Coding | N-terminal RenG luciferase fragment |
| 3 | BBa_25CVCYGH | C'-RenG luciferase | Coding | C-terminal RenG luciferase fragment |
| 4 | BBa_25PTH3PS | spytag | Coding (peptide) | Short peptide binding SpyCatcher |
| 5 | BBa_257J1PEJ | spycatcher002_design33_n29 | Coding | Engineered SpyCatcher mutant 33_n29 |
| 6 | BBa_25E2PO9G | spycatcher002_design17_n22 | Coding | Engineered SpyCatcher mutant 17_n22 |
| 7 | BBa_25F2XK6E | spycatcher002_design0_n15 | Coding | Engineered SpyCatcher mutant 0_n15 |
| 8 | BBa_25CJBY99 | spycatcher002_design0_n0 | Coding | Engineered SpyCatcher mutant 0_n0 |
| 9 | BBa_25NYARGM | spycatcher002_design0_n1 | Coding | Engineered SpyCatcher mutant 0_n1 |
| 10 | BBa_25AF6R01 | spycatcher002_design0_n13 | Coding | Engineered SpyCatcher mutant 0_n13 |
| 11 | BBa_25VI00F2 | spycatcher_35 | Coding | Engineered SpyCatcher mutant 35 |
| 12 | BBa_25CMQ82K | spycatcher_29 | Coding | Engineered SpyCatcher mutant 29 |
| 13 | BBa_25DJ0CFO | SpyCatcher factory(N'-RenG luciferase+linker*2+SpyCatcher+linker+his-tag) | Device | Fusion: N'-RenG + SpyCatcher(original SpyCatcher without mutation)+ His-tag, to generate SpyCatcher |
| 14 | BBa_257ZSVJ2 | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher002_design33_n29+linker+his-tag) | Device | Fusion: N'-RenG + mutant 33_n29, to generate mutant SpyCatcher |
| 15 | BBa_25VCRFHH | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher002_design17_n22+linker+his-tag) | Device | Fusion: N'-RenG + mutant 17_n22, to generate mutant SpyCatcher |
| 16 | BBa_25XT1GPI | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher002_design0_n15+linker+his-tag) | Device | Fusion: N'-RenG + mutant 0_n15, to generate mutant SpyCatcher |
| 17 | BBa_257IGSFX | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher002_design0_n0+linker+his-tag) | Device | Fusion: N'-RenG + mutant 0_n0, to generate mutant SpyCatcher |
| 18 | BBa_2572RS9F | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher002_design0_n1+linker+his-tag) | Device | Fusion: N'-RenG + mutant 0_n1, to generate mutant SpyCatcher |
| 19 | BBa_25FGZXFN | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher002_design0_n13+linker+his-tag) | Device | Fusion: N'-RenG + mutant 0_n13, to generate mutant SpyCatcher |
| 20 | BBa_25UKKRTG | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher_35+linker+his-tag) | Device | Fusion: N'-RenG + mutant catcher35, to generate mutant SpyCatcher |
| 21 | BBa_255VR1P8 | SpyCatcher factory(N'-RenG luciferase+linker*2+spycatcher_29+linker+his-tag) | Device | Fusion: N'-RenG + mutant catcher29, to generate mutant SpyCatcher |
| 22 | BBa_250WYFV2 | SpyCatcher factory-no luciferase | Device | SpyCatcher fusion without RenG, to generate original SpyCatcher |
| 23 | BBa_K1223006 | SpyCatcher factory(C'-RenG luciferase+linker*2+SpyCatcher+linker+his-tag) | Device | Fusion: C'-RenG + SpyCatcher(original SpyCatcher without mutation)+ His-tag, to generate SpyCatcher |
| 24 | BBa_25YF37M3 | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher002_design33_n29+linker+his-tag) | Device | Fusion: C'-RenG + mutant 33_n29, to generate mutant SpyCatcher |
| 25 | BBa_25VK4TV3 | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher002_design17_n22+linker+his-tag) | Device | Fusion: C'-RenG + mutant 17_n22, to generate mutant SpyCatcher |
| 26 | BBa_25LWT3SZ | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher002_design0_n15+linker+his-tag) | Device | Fusion: C'-RenG + mutant 0_n15, to generate mutant SpyCatcher |
| 27 | BBa_25C8WSRP | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher002_design0_n0+linker+his-tag) | Device | Fusion: C'-RenG + mutant 0_n0, to generate mutant SpyCatcher |
| 28 | BBa_25GH6TMN | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher002_design0_n1+linker+his-tag) | Device | Fusion: C'-RenG + mutant 0_n1, to generate mutant SpyCatcher |
| 29 | BBa_25PG0NE0 | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher002_design0_n13+linker+his-tag) | Device | Fusion: C'-RenG + mutant 0_n13, to generate mutant SpyCatcher |
| 30 | BBa_25YGME6B | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher_35+linker+his-tag) | Device | Fusion: C'-RenG + mutant 35, to generate mutant SpyCatcher |
| 31 | BBa_259D22HL | SpyCatcher factory(C'-RenG luciferase+linker*2+spycatcher_29+linker+his-tag) | Device | Fusion: C'-RenG + mutant 29, to generate mutant SpyCatcher |
| 32 | BBa_25NAGCBQ | pET-IDT_tag factory(C'-RenG luciferase) | Device | C'-RenG luciferase in pET-IDT, to generate SpyTag |
| 33 | BBa_256X9XUM | pET-IDT_tag factory(N'-RenG luciferase) | Device | N'-RenG luciferase in pET-IDT, to generate SpyTag |
Experiment C
This experiment aims to build a modular SpyCatcher assembly system that divides the SpyCatcher gene into multiple overlapping segments, allowing selective mutagenesis within specific regions while keeping the rest of the sequence constant.
Each segment is designed with overlapping ends for seamless assembly and precise control of mutation sites.
Segment-1 (either N′-RenG or C′-RenG luciferase fusion, BBa_25W9IAN8, BBa_25BRXOP8) covers SpyCatcher 1–136 bp and includes protective bases, an XbaI restriction site, RBS, and a spacer. This region is fixed and not mutated, ensuring correct expression and fusion with luciferase fragments.
Segment-2 (118–180 bp, BBa_25S6NORT) and Segment-4 (253–318 bp, BBa_2567CEWK) are designated mutagenic regions, allowing targeted sequence diversification.
Segment-3 (161–271 bp, BBa_25BF7YWR) and Segment-5 (299–318 bp + BlpI site, BBa_259BBYRK) remain unmutated, serving as structural anchors for stable reassembly.
By combining these fragments through overlap extension, the full-length SpyCatcher can be reconstructed with mutations confined to specific domains, minimizing disruption to essential folding regions.
This design enables controlled evolution and structural analysis of SpyCatcher variants, supporting the identification of key residues that influence binding kinetics and stability while maintaining overall structural integrity.
Figure 3. Experiment C parts design schematic diagram.
Created with BioRender.
| Item | Number | Name | Type | Description |
|---|---|---|---|---|
| 1 | BBa_25M1PVNB | BlpI | Restriction site | Type IIS restriction enzyme site |
| 2 | BBa_25W9IAN8 | SpyCatcher segment-1(protective bases + XbaI+RBS+spacer+N'-RenG luciferase+Spycatcher 1-136 bp) | Device | N'-RenG luciferase + SpyCatcher 1–136 bp, which can't be mutate |
| 3 | BBa_25BRXOP8 | SpyCatcher segment-1(XbaI+RBS+spacer+C'-RenG luciferase+Spycatcher 1-136 bp) | Device | C'-RenG luciferase + SpyCatcher 1–136 bp, which can't be mutate |
| 4 | BBa_25S6NORT | SpyCatcher segment-2(Spycatcher 118-180 bp) | Device | SpyCatcher fragment 118–180 bp, which can be mutate |
| 5 | BBa_25BF7YWR | SpyCatcher segment-3(Spycatcher 161-271bp) | Device | SpyCatcher fragment 161–271 bp, which can't be mutate |
| 6 | BBa_2567CEWK | SpyCatcher segment-4(Spycatcher 253-318 bp) | Device | SpyCatcher fragment 253–318 bp, which can be mutate |
| 7 | BBa_259BBYRK | SpyCatcher segment-5(Spycatcher 299-318 bp+BlpI+protective bases) | Device | SpyCatcher fragment 299–318 bp + BlpI, which can't be mutate |