Parts
Parts
Overview
This year, BJWZ-China has made contributions of 2 suceed basic parts, 4 composite parts and 2 biocontainment system. For the composite parts, the team has engineered comprehensive genetic circuits comprising operators, distinct ribosome binding sites (RBS), and signal peptides， linkers, which are designed to enable direct utilization by future iGEM participants. Additionally, a suicidal system derived from BNU-China and BJWZ-China-2024 has been integrated to enhance the biosafety of the engineered system, thereby mitigating the potential risks of genetically modified organisms impacting natural ecosystems.
Basic Part
This year, BJWZ-China targeted PFAS pollution using DhaA dehalogenase. After reviewing recent studies and comparing dehalogenases, DhaA was chosen for its compatibility with chassis strains, broad substrate range, high stability, and engineering potential. Its advantages—including computational design, chemical modification, and AI-optimized advances—have reshaped enzymatic catalysis research. Integrating synthetic biology and metabolic engineering, DhaA is poised to drive breakthroughs in PFAS bioremediation and green synthesis. Additionally, the fluoride ions released from PFAS are transported out of the cells by fluoride ion transporters, thereby enhancing the toxicity tolerance and treatment efficiency of engineered bacteria.
| Registry Code | Part Name | Profile | Advantages/ Disadvantages | Status | Length(bp) |
|---|---|---|---|---|---|
| BBa_25TW5KB0 | dhaA | Haloalkane dehalogenase, which from Mycobacterium tuberculosis H37Rv is a two-domain protein having a catalytic domain of an alpha/beta hydrolase fold and a cap domain. The active site residues and the halide-stabilizing residues have been identified as Asp109, Glu133, His273, Asn39 and Trp110. | Broad substrate spectrum, high thermal stability, strong modifiability, and adjustable stereoselectivity. | Succeed | 903 |
| BBa_25PJZ648 | Rv2296 | Derived from Mycobacterium tuberculosis, belongs to the α/β-hydrolase family and catalyzes the dehalogenation of short-chain haloalkanes by cleaving carbon-halogen bonds via an SN2 mechanism. It potentially contributes to pathogen resistance against host-derived halogenated metabolites and holds promise for environmental decontamination of halogenated pollutants. | Its recombinant expression often leads to inclusion body formation, and the active site and catalytic mechanism remain to be validated. | In-Fusion is failed | 903 |
| BBa_250RQMKY | Rv1833c | Derived from Mycobacterium tuberculosis H37Rv, is one of the genes encoding a putative dehalogenase, which is speculated to participate in the hydrolytic dehalogenation reaction of haloalkanes. | Its individual function is not clear, its dehalogenation activity lacks verification through pure enzyme experiments, and it needs to rely on the combined application of multiple antigens. | Repeat electrophoresis | 861 |
| BBa_25E9O5TI | CrcB | Derived from Escherichia coli str. K-12 substr. MG1655. It functions as a fluoride ion transporter (or channel), which can rapidly export intracellular fluoride ions. | With the advantages of reducing intracellular fluoride concentration, alleviating fluoride toxicity, and enhancing the fluoride tolerance of microorganisms. | Succeed | 384 |
Composite Part
This year, BJWZ-China assembled several new Composite Parts that serve as core modules for constructing genetic devices in our project design. These modules can achieve specific functions such as gene expression regulation, metabolic pathway mediation, and biosensing. By modularly combining functional elements, they provide flexible components to achieve our project goals, enabling the development of complex biological functions in our engineered strain.
| Registry Code | Part Name | Profile | Allocation | Status | Length(bp) |
|---|---|---|---|---|---|
| BBa_25ZMTQVL | prmA-RBS | PFAS-sensitive promoters and ribosome binding sites with high expression requirements | 1-174 bp: prmA 223-248 bp: RBS Other sequence: Intergenic Sequence. | Succeed | 248 |
| BBa_25115VG9 | dhaA-GFP | Dehalogenase and reporter protein | 1-900 bp: dhaA 901-945 bp: 3×(GGGGS) Linker; Flexible linker sequence, often used in the construction of fusion proteins to connect different functional domains 946-963 bp: 6×His; A tag for the purification of protein which focus in the production of recombinant proteins 964-1020 bp: P2A; Self-cleaving peptide, When the fusion gene is expressed as 'Protein A-P2A-Protein B', it produces independent 'Protein A' and 'Protein B' after translation, without the need for additional cleavage enzymes. 1027-1740 bp: sfGFP；Superfolder GFP is a variant of GFP engineered through molecular engineering. It exhibits higher brightness than wild-type GFP and can be detected with higher sensitivity. Other sequence: Intergenic Sequence. | Succeed | 1740 |
| BBa_25S5Y6E4 | T7-F(-) riboswitch-RBS | Fluoride-responsive riboswitch mediates conditional initiation of downstream gene expression. | 1-19 bp: T7 promoter; Used to regulate gene expression of recombinant proteins. 20-34 bp: Intergenic Sequence. 35-112 bp: 78 Psy F Riboswitch（ncRNA）; As an RNA regulatory element, it controls the expression of downstream gene(s) by directly binding fluoride ions (F⁻). At low fluoride concentrations, the riboswitch adopts a "closed" conformation, thereby preventing the transcription or translation of the downstream gene(s). When fluoride concentration increases, F⁻ binds directly to the aptamer domain of the riboswitch, inducing a conformational change that derepresses and thereby initiates the expression of the downstream gene(s). 123-131 bp: RBS BBa_B0034 | Succeed | 131 |
| BBa_259J06HO | CrcB-mCherry | Fluoride ion transporter and reporter gene. | 1-381 bp: crcB 382-426 bp: 3× (GGGGS) Linker; 427-444 bp: 6×His; 445-501 bp: P2A; 508-1218 bp: mCherry; A widely used red fluorescent protein (RFP), mCherry is derived from the coral Discosoma striata and exists as a monomeric fluorescent protein. It is applied for visual monitoring of gene expression (e.g., promoter activity, transcriptional efficiency) and serves as an output signal in genetic circuits. 1219-1305 bp: rrnB T1 terminator; Terminates transcription. Other sequence: Intergenic Sequence. | Succeed | 1305 |
Biocontainment system
Since a type II antitoxin-toxin (TA) system is functioning effectively as a suicidal system for killing our manufactured leakage microorganisms, we were inspired by last year’s contribution and build the exact same sequence pathway using the parts listed below.
| Registry Code | Part Name | Profile | Allocation | Status | Length(bp) |
|---|---|---|---|---|---|
| BBa_25F1YN2E | Lac-MazE/F | Lac operon-regulated MazE/F toxin-antitoxin protein system for engineered bacterial suicide | 1-19 bp: T7 promoter; 20-44 bp: Lac oprator; Used to control gene expression by lactose. 59-304 bp: MazE; MazE is a key component of the widely distributed Toxin-Antitoxin System (TA) in bacteria, typically forming a functional pair with the toxin protein MazF. As the antitoxin of the type II TA system, MazE prevents cell death by directly binding to the toxin MazF and inhibiting its activity. 305-316 bp: GS Linker; 317-334 bp: 6×His; 348-359 bp: RBS; 366-701 bp: MazF; MazF is the core toxin protein of the classical toxin–antitoxin (TA) system in bacteria, which forms a functional pair with the antitoxin MazE and plays a key role in cellular stress responses and gene regulation. In normal conditions, MazF is bound by MazE at a 1:1 ratio, forming an inactive MazE–MazF complex that maintains cell viability. Upon exposure to nutrient deprivation, oxidative stress, or antibiotics, MazE is degraded by proteases such as ClpAP and Lon, thereby releasing the active form of MazF. 710-737 bp: rrnB T2 terminator. Other sequence: Intergenic Sequence. | Failed to function | 737 |
| BBa_25T7DKZH | T7-RBS-MazE/F | MazE/F toxin-antitoxin protein system for engineered bacterial suicide that co-regulated by the T7 promoter and a 37°C temperature-sensitive RBS. | 1-19 bp: T7 promoter; 20-61 bp: RBS; RNA thermometer to prevent environment leakage of our engineered bacteria. When it is 37℃, the stem-ring structure of is the RNA thermometer opened, thus, RBS is exposed, the MazE(antitoxin protein) could be normally expressed and can bind MazF(toxin protein) protein makes MazF ineffective. 70-315 bp: MazE; 316-327 bp: GS Linker; 328-345 bp: 6×His; 359-370 bp: RBS; 377-712 bp: MazF; 721-748 bp: rrnB T2 terminator. Other sequence: Intergenic Sequence. | Succeed; However, in our engineered bacteria, leaky expression occurred, and no transformants were obtained. | 748 |
| BBa_25Y0Q84D | araC-ccdB | The kill switch controlled by L-arabinose | 1-28 bp: T7 terminator； 32-910 bp: araC; AraC represses at araO1 but activates pBAD with L-arabinose, initiating ccdB. 1056-1221 bp: araBAD promoter; In the absence of L-arabinose, AraC binds as a monomer to araO1 and araI1, forming a DNA loop that inhibits RNA polymerase binding to the pBAD promoter, thereby turning off transcription with minimal leaky expression. In the presence of L-arabinose, the sugar binds to AraC, causing it to adopt a dimeric conformation that binds to araI1 and araI2, recruits RNA polymerase, and initiates high-level transcription. 1253-1558 bp: ccdB; CcdB inhibits DNA replication without lysis, sparing native microbes. Soil arabinose prevents engineered bacteria leakage. 1563-1649 bp: rrnB T1 terminator. Other sequence: Intergenic Sequence. | Succeed | 1649 |