Parts
Overview
SCU-China used 28 basic components and 26 composite components in this year's work in the wet laboratory, among which 17 basic components and 26 composite components were newly registered.
These components play roles in multiple circuits: responsible for knocking out the endogenous tasA and sinR in Bacillus subtilis, remodeling TasA to enhance adhesion to polystyrene, sensing and adsorbing cadmium ions, and a density-dependent suicide switch circuit.
Highlight
Among all of our parts, the most noteworthy is our use of Bacillus subtilis surface display technology, where we transformed and applied Bacillus subtilis to fuse with various binding proteins, enabling it to adhere to solid substrates in wastewater under diverse conditions and providing sustainable solutions for wastewater treatment.
During the design process of TasA-binding protein, we found that directly constructing large fusion proteins has significant limitations: such proteins may negatively affect TasA secretion function and self-assembly ability, both of which are crucial factors determining the adhesion performance of TasAnchor, ultimately leading to reduced adhesion capability. We proposed using the SpyTag-SpyCatcher system to redesign the TasA protein, constructing TasA-SpyTag, and achieving material adhesion through binding protein-SpyCatcher.
|
To prevent engineered bacteria from leaking into the environment, we designed a density-dependent suicide switch circuit. This circuit incorporates the bacteria's endogenous quorum sensing system and uses toxin/antitoxin antagonism, allowing engineered bacteria to survive at high densities when adhered to a material, but causing them to die at low densities when detached from the material surface. This circuit provides a new idea for other iGEM teams to ensure biosafety.
|
Basic Part
| Parts No. | Parts Name | Description |
|---|---|---|
| BBa_25358K38 | PS-tag1(new) | PS tag is a short peptide derived from deep learning that specifically binds to polystyrene. |
| BBa_25YX41PK | PS-tag2(new) | PS tag is a short peptide derived from deep learning that specifically binds to polystyrene. |
| BBa_25MOXUQX | Mfp5(new) | Mfp5 is a key protein in mussel byssal plaques that mediates strong underwater adhesion to various surfaces. |
| BBa_251KOVKJ | SpyTag(new) | A short peptide that can form a stable isopeptide bond with SpyCatcher through specific interactions. |
| BBa_25YSN8RV | SpyCatcher(new) | The SpyTag-SpyCatcher system is a highly efficient covalent bioconjugation tool, whose core function lies in forming stable isopeptide bonds through specific interactions between a short peptide (SpyTag) and a protein (SpyCatcher). A protein that can form a stable isopeptide bond with SpyTag through specific interactions. |
| BBa_25L81IR9 | His-tag(new) | His tag is a tag used for protein purification. |
| BBa_2591VE13 | TasA(new) | TasA is a major biofilm protein of Bacillus subtilis with self-assembly ability, forming adhesive fibrous structures in the extracellular matrix. |
| BBa_25CDGN68 | pHpaII(new) | The pHpall promoter is a pH-responsive regulatory element that modulates gene expression under different environmental pH conditions. |
| BBa_25O00TCO | linker(TasA-binding protein)(new) | A linker links TasA and binding protein |
| BBa_250NOJGV | Bacillus subtilis RBS (in pHT01)(new) | RBS in pHT01 plasmid |
| BBa_25S3H0RP | Linker (tasA-smtA)(new) | A linker links TasA and SmtA |
| BBa_25JBQTIE | pJOE8999(new) | pJOE8999 is a CRISPR-Cas9 gene editing shuttle plasmid. Cas9 and sgRNA were expressed under the induction of mannose, and the target gene was knocked out or replaced. The plasmid was lost at 37 °C and the traceless editing was completed. |
| BBa_259N689W | smtA(new) | A gene encoding a metallothionein-like protein whose expression product is capable of binding heavy-metal ions. |
| BBa_25FX33M1 | cadR(new) | The protein encoded by cadR belongs to the MerR family of transcription factors, which can sense metal ions such as Cd ( II ), Zn ( II ), and Hg ( II ), and activate the expression of downstream genes. |
| BBa_253O82GY | tasA-sinR-sgRNA1(new) | A sgRNA targeting sinR-tasA |
| BBa_254U614K | tasA-sinR-sgRNA2(new) | A sgRNA targeting sinR-tasA |
| BBa_25EFY2KM | tasA-sinR-knockout homologous sequence(new) | tasA-sinR-knockout homologous sequence |
| BBa_K5432030 | pT7-lacO | A strong T7 promoter regulated by the LacI repressor, enabling IPTG-inducible high-level gene expression in E. coli systems. |
| BBa_K1628202 | pGrac | A hybrid IPTG-inducible promoter regulated by LacI. |
| BBa_K2142001 | mazE | The antitoxin component of the MazEF toxin–antitoxin system. |
| BBa_K302033 | mazF | The toxin component of the MazEF system. |
| BBa_K1088018 | lacI | Encodes the LacI repressor protein that binds to the lac operator to suppress transcription from LacI-regulated promoters. |
| BBa_K2273003 | ComX | A signal peptide precursor in the ComQXPA quorum-sensing system of Bacillus subtilis. |
| BBa_2508ZG2U | mcherry | A red fluorescent protein. |
| BBa_K143013 | p43 | A constitutive promoter from Bacillus subtilis. |
| BBa_K2273000 | psrfA | A quorum-sensing–responsive promoter activated by the ComA–ComP two-component system in Bacillus subtilis. |
| BBa_K4934022 | trp-terminator | The trp terminator efficiently halts transcription downstream of the trp operon, preventing unwanted transcriptional read-through. |
| BBa_2509EZ6U | Fd terminator | central terminator from bacteriophage fd |
Composite Part
| Parts No. | Parts Name | Description |
|---|---|---|
| BBa_25IBB20N | Surface display module of Bacillus subtilis immobilization (new)(Awards) | TasA is a major biofilm protein of Bacillus subtilis with self-assembly capability, forming adhesive fibrillar structures in the extracellular matrix. We use it as a foundation for constructing our other fusion proteins. |
| BBa_25FF85T9 | psrfA-mcherry(new) | A reporter construct in which mCherry expression is driven by the quorum-sensing promoter psrfA to monitor density-dependent activity. |
| BBa_25JG7VXG | pGrac-mazF(new) | An inducible toxin expression module where the mazF gene is driven by the IPTG-regulated promoter pGrac, enabling controllable cell death. |
| BBa_259Z0CT1 | pgrac-mazF-p43-mazE(new) | A toxin–antitoxin module integrating inducible mazF expression with constitutive mazE production for balanced biosafety control. |
| BBa_25885P5M | psrfA-pgrac-mazF-p43-mazE(new) | A quorum-sensing–regulated density-dependent suicide system combining psrfA control with the pGrac–mazF–p43–mazE toxin–antitoxin circuit to ensure biosafety in Bacillus subtilis. |
| BBa_25JYVPQN | pGrac-his-mcherry(new) | Directly ligate mCherry into the plasmid backbone to verify whether fluorescence is expressed normally. |
| BBa_253W83T4 | pGrac-tasA-linker-mfp5(new) | We constructed a TasA-Mfp5 fusion protein, aiming to enhance the adhesion efficiency of engineered bacteria by leveraging Mfp5's sub-surface adhesion capability. |
| BBa_25B4WQNJ | pGrac-tasA-linker-pstag1(new) | We constructed a TasA-PS tag fusion protein, hoping our engineered bacteria would stably adhere to the polystyrene surface. |
| BBa_25UC8F9U | pGrac-tasA-linker-pstag2(new) | We constructed a TasA-PS tag fusion protein, hoping our engineered bacteria would stably adhere to the polystyrene surface. |
| BBa_25J8NJTQ | pGrac-tasA-linker-SpyTag(new) | Acting in concert with the spycatcher fusion protein, it enables bacterial strains to bind to it via the spytag, thereby adhering to surfaces coated with the spycatcher fusion protein. |
| BBa_25XOUXNV | pGrac-tasA-linker-mcherry(new) | We aim to evaluate the expression efficiency of our designed system by detecting the fluorescence of mCherry. |
| BBa_25USBKHP | pHpaII-mcherry(new) | Directly ligate mCherry into the plasmid backbone to verify whether fluorescence is expressed normally. |
| BBa_251MS0E1 | pHpaII-tasA(new) | TasA is a major biofilm protein of Bacillus subtilis with self-assembly ability, forming adhesive fibrous structures in the extracellular matrix. |
| BBa_25NU84IF | pHpaII-tasA-linker-mfp5(new) | We constructed a TasA-Mfp5 fusion protein, aiming to enhance the adhesion efficiency of engineered bacteria by leveraging Mfp5's sub-surface adhesion capability. |
| BBa_252OQ23N | pHpaII-tasA-linker-pstag1(new) | We constructed a TasA-PS tag fusion protein, hoping our engineered bacteria would stably adhere to the polystyrene surface. |
| BBa_25S5TQ4M | pHpaII-tasA-linker-pstag2(new) | We constructed a TasA-PS tag fusion protein, hoping our engineered bacteria would stably adhere to the polystyrene surface. |
| BBa_256MR4QL | pHpaII-tasA-linker-SpyTag(new) | Acting in concert with the spycatcher fusion protein, it enables bacterial strains to bind to it via the spytag, thereby adhering to surfaces coated with the spycatcher fusion protein. |
| BBa_25Y3JODM | pHpaII-tasA-linker-mcherry(new) | We aim to evaluate the expression efficiency of our designed system by detecting the fluorescence of mCherry. |
| BBa_25DYUWZC | pT7-SpyCatcher-mcherry-histag(new) | We aim to evaluate the expression efficiency of our designed system by detecting the fluorescence of mCherry. His tag enables us to purify and obtain this fusion protein. |
| BBa_25I514B7 | pT7-SpyCatcher-mfp5-histag(new) | We constructed a TasA-Mfp5 fusion protein, aiming to enhance the adhesion efficiency of engineered bacteria by leveraging Mfp5's sub-surface adhesion capability. His tag enables us to purify and obtain this fusion protein. |
| BBa_250E313T | pGrac-tasA-smtA(new) | Under the induction of IPTG，it can express the fusion protein constructed by metallothionein-like protein SmtA and Bacillus subtilis biofilm-forming protein TasA in Bacillus subtilis. |
| BBa_256QQOKX | pHpaII-tasA-smtA(new) | It can continuously express the fusion protein constructed by metallothionein-like protein Smt A and Bacillus subtilis biofilm forming protein Tas A in Bacillus subtilis. |
| BBa_2528UY61 | pGrac-CadR-pCadR-mcherry(new) | Under the induction of IPTG，it can specifically recognize cadmium ions and inducing the expression of the red fluorescent protein mCherry. |
| BBa_25NLGQQY | pHpaII-CadR-pCadR-mcherry(new) | It can specifically recognize cadmium ions and inducing the expression of the red fluorescent protein mCherry. |
| BBa_25XRIBW0 | pHpaII-tasA-smtA-p43-tasA-mfp5(new) | A fusion protein capable of adsorbing heavy metal ions was produced and bound to the attachment protein on the biofilm of Bacillus subtilis. |
| BBa_25A85ZC3 | pGrac-CadR-pCadR-mcherry-p43-tasA-mfp5(new) | Under the induction of IPTG, it can sense heavy metals in the surrounding environment and produce fluorescent protein to characterize, and bind to the attachment protein on the biofilm of Bacillus subtilis. |