Parts
Title


During this project, we designed, engineered, assembled, and rigorously assessed a series of novel parts, 17 basic and 8 composite, alongside 16 established parts. With a various selection of parts and the primers we designed, it allowed us to construct and validate our three different biological circuits.

Best New Basic Part

Our newly introduced basic part, PflQ2MT (BBa_25VCEOE5), encodes the PflQ2 metallothionein. For surface display, this metallothionein is fused to the Lpp-OmpA membrane anchor (BBa_K1694010), integrating it into our Main Circuit responsible for specific cadmium sequestration. The efficacy of this system relies on the metallothionein possessing high specificity and exceptional efficiency for cadmium ion binding. The characterization and validation of this surface-expressed binding domain significantly contributes to the functional metal-binding protein library, providing valuable insights for the iGEM and synthetic biology community.
For the detailed experimental and simulation results of our Best New Basic Part, please refer to our Wet Lab and Dry Lab pages.

Basic Parts

We utilized a diverse collection of pre-existing and newly developed basic parts, including promoters, coding sequences, and terminators, regular and irregular, to construct our genetic circuit. These foundational elements were essential for controlling gene expression and ensuring the functional integrity of our design. The details of these basic parts are provided below.

Type Parts No. Part Name Description
Existing Parts
Promoter BBa_K4278705 EM7 promoter EM7 promoter, a derivation from T7 promoter, intermediate expression, widely used in P.putida KT2440
BBa_K5291021 PopdH PopdH promoter from P. aeruginosa with capability of sensing citrate concentration. Act as the sensor in the citrate-dependent kill switch.
BBa_K2839070 PLtetO-1 Promoter regulated by the Tet repressor protein (TetR) and anhydrotetracycline. As a regulatory tuning input for the cadmium biosensor.
BBa_R0051 Plambda cI Promoter derived from bacteriophage Lambda that is negatively regulated by the Lambda cI repressor protein. Associate with its repressor for a not gate in the kill switch.
RBS BBa_B0034 RBS (Elowitz 1999) A well-characterized RBS originating from the Elowitz repressilator.
Coding BBa_K1694010 Lpp-OmpA Lpp-OmpA is a chimeric protein, where Lpp is a signal peptide and OmpA is a membrane protein for anchorage on bacteria membranes. It was used for displaying cadmium binding protein on the surface of engineered bacteria.
BBa_K2152003 Phi X 174 Lysis Protein E Phi X 174 Lysis Protein E is a bacteriophage protein that induces host cell lysis and executes the kill switch.
BBa_K3257071 LVA Degradation Tag A short peptide sequence that marks proteins for rapid degradation by bacterial proteases such as ClpAP and ClpXP, for fast replanshipment in the cadmium biosensor.
BBa_J18918 TEV Cleavage Site A specific recognition sequence for the TEV protease, for verification of the surface expression and having potential for extension of the concept.
Regulatory BBa_C0051 Lambda cI Repressor A transcriptional repressor protein from bacteriophage Lambda. Repressing respective promoter for a not gate in the kill switch.
BBa_K2621015 RiboSwitch Trigger RiboSwitch trigger sequence which will open RiboSwitch aptamer and proceed translation.
Reporter BBa_I746916 sfGFP An enhanced variant of GFP known for its robust and rapid folding, even under challenging conditions. For monitoring the expression in kill switch.
BBa_K3781015 mVenus mVenus is a monomeric yellow fluorescent protein (YFP) derived from Aequorea victoria GFP. For monitoring the expression in kill switch.
Terminator BBa_K3257021 Lambda T0 A strong transcriptional terminator derived from bacteriophage Lambda.
BBa_K864601 Lambda T1 A strong transcriptional terminator from bacteriophage Lambda.
New Parts
Promoter BBa_25CAR3OY Pcad Promoter regulated by the CadR protein in response to cadmium. Act as the “kit” for the cadmium biosensor.
BBa_25JN1UBJ P14g A strong constitutive promoter often used for high-level expression.
RBS BBa_25TMR0E8 pJL1-sfGFP Strong P. putida ribosome binding site. (yes this is a RBS)
BBa_25W6KHRF RBS 12 Moderate P.putida ribosome binding site.
BBa_25KUISST RBS 14 Very weak P. putida ribosome binding site.
BBa_2547RBWN RBS 15 Moderate P. putida ribosome binding site.
Coding BBa_25VCEOE5 PflQ2MT PflQ2MT is the coding sequence for a metallothionein protein from P. fluorescens, whose primary function is cadmium binding, and is the cadmium binding protein that we used in our main circuit.
BBa_25WWG3IU EC20 EC20 is a synthetic phytochelatin, a cysteine-rich peptide with high affinity for cadmium. Codon optimized and designed for synthesis to minimise complexity index.
BBa_25164G5C 6x His Tag A common affinity tag consisting of six histidine residues for purification and detection of attached proteins. Valine is added in the front and end of the tag as spacer.
BBa_25RWHTND GS Linker Flexible polypeptide linkers made of glycine and serine repeats, allowing for independent folding while still maintaining independent activity.
Regulatory BBa_251WLXJ9 cadR (P. putida Codon Optimized) A transcriptional regulator from P. putida that exhibits an ultraspecific response to cadmium ions and activates Pcad promoter, acts as the detector in our biosensor.
BBa_259S6D4J tetR (P. putida Codon Optimized) A well-known transcriptional repressor that binds and represses PLtetO-1, acts as the tuning input of the biosensor.
BBa_25JZRMKG tctD (Origin P. putida) A coding sequence found in P. putida who is involved in the TCA (tricarbonxyl acid) cycle, a regulatory protein in the tctD-tctE two component system. Acts as a tricarbonxyl acid (such as citric acid) sensing protein that represses PopdH with citrate acid absence.
BBa_25S6BMR8 RiboSwitch Aptamer RiboSwitch aptamer with hidden RBS (BBa_B0034), open once trigger RNA binds and proceed translation.
Reporter BBa_25IVGUN2 mScarlet-I3-NCwt A variant of the red fluorescent protein mScarlet, acts as the reporter of our biosensor and the indicator of the expression in our kill switch.
Terminator BBa_25RQDKM3 eT1 A functional transcriptional terminator.
BBa_25WWU8MU eT7 A transcriptional terminator specifically recognized by T7 RNA polymerase.

Composite Parts

We utilized and engineered a variety of composite parts to construct our system. These included three primary functional devices: the main circuit responsible for cadmium binding, a dedicated biosensor device, and an essential genetic kill switch. These composite parts integrate multiple basic parts to achieve complex biological functions.

Parts No. Part Name Description
BBa_B0015 Double Terminator (B0010-B0012) A composite part consisting of two strong transcriptional terminators in tandem.
BBa_25OUGOXH Double Terminator (Lambda T1 - eT7) A composite part consisting of two strong transcriptional terminators in tandem.
BBa_25PJ1C5O Lpp-Ompa-6G-TEV Cleavage Site A universal composite part for surface expression of the binding proteins, with protease cleavage site.
BBa_25OWO9RF Surface Expression of metallothionein for E. coli Main Circuit for surface expression of metallothionein, specified in E. coli.
BBa_25R6AJ4F Surface Expression of metallothionein for P. putida Main Circuit for surface expression of metallothionein, specified in P. putida.
BBa_25310AKD Surface Expression of EC20 for P. putida Main Circuit for surface expression of EC20, specified in P. putida.
BBa_256ORVBQ RiboSwitch Cadmium Biosensor for P. putida Cadmium biosensor with RiboSwitch, aiming at glowing with cadmium persistence, with fast replenishment.
BBa_25VVGSTQ Simplified RiboSwitch Cadmium Biosensor for P. putida Simplified version of the cadmium biosensor with RiboSwitch.
BBa_25CTXIKQ Citrate Kill Switch for P. putida P. putida kill switch under the absence of citrate.

Part Collection

We developed a specialized RiboSwitch Part Collection, which offers a powerful option for post-transcriptional regulatory control of gene expression.

RiboSwitch
The Riboswitch is fundamentally based on the Toehold Switch architecture. In its OFF state, the RBS is sequestered within a hairpin structure formed by the aptamer, preventing ribosomal binding. Upon the binding of a specific trigger RNA, which is transcribed from a separate regulatory factor, the structure undergoes a conformational change that opens the aptamer, exposing the RBS and allowing the ribosome to bind and initiate downstream translation.
Part No. Part Name Description
BBa_25S6BMR8 Strong RBS - RiboSwitch Aptamer RiboSwitch aptamer with hidden RBS (BBa_B0034), open once trigger RNA binds and proceed translation.
BBa_K2621015 Strong RBS - RiboSwitch Trigger RiboSwitch trigger sequence which will open Riboswitch aptamer and proceed translation.

Plasmids and Backbones +

We selected and utilized a range of plasmid backbones, carefully chosen based on the host chassis, different circuit, and the expression level. We used plasmids with different copy numbers to optimize the expression of distinct circuits and with different antibiotic resistance to enable clear selection. The specific details of these plasmids are listed below.

Primers +

We designed a comprehensive set of primers tailored for various applications, including assembly, colony PCR, and amplification. A detailed list of the designed primers is available below.

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