Parts Overview
This year, we designed and constructed our project and our parts based on protein-protein interactions, including rearrangements, combinations, and cleavages. Through these parts, we constructed a cell-free rapid detection system comprising a dual-input AND logic circuit and a positive feedback pathway.
Our collection includes:
- Basic parts: coiled-coil domains, fragments of the COI1-JAZ1 pathway, novel and improved proteases and reporter proteins;
- Composite parts: the proteins for the Input-AND Gate-Output circuit design, and several fusion proteins for experimental verification.
A series of coiled-coils domains fused with downstream functional protein domains is what features our project the most. Specific coiled-coil domains can form pairs, resulting in heterodimers. With the rearrangement of the coiled-coil domains, split proteins connected to them can be brought into proximity and regain function as a reconstituted protein. This mechanism forms the basis of our project system, enabling us to achieve our AND gate effect.
To prevent unexpected rearrangement and control the pairing of coiled-coil domains, we utilized proteases and their corresponding cleavage sites to achieve this process. Based on this idea, we also selected protease AvrRpt2 as our detection target and input signal, and built a positive feedback loop to increase the input signal.
COI1-JAZ1 binding complex is also included as the receptor of one of our inputs. Additionally, split trehalase, luciferase, and green fluorescent protein are used for project output. We optimized these split signal proteins and linked them to the coiled-coil domains, connecting them to our processing circuit and producing detectable outputs.
By combining these parts, we constructed a proteolysis and coiled-coil-based rapid detection system with an Input-AND Gate-Output process. AvrRpt2 and phytotoxin COR serve as Input signals. Coiled-coil domains and proteases induce the AND Gate process. Three split reporters are selected for downstream Output and are available for detection.
Besides the parts of the project pathway, we also included several parts designed for experiment verification. Basic coding parts are composed in a certain sequence to express specific fusion proteins that can be used to prove the function of certain basic parts separately. We used these fusion proteins to verify the function of coiled-coil domains, the cleavage activity of AvrRpt2, and the intensity of protein interactions. We hope this design strategy could be an inspiration for future protein-based systems.
The detailed list of our Basic Parts and Composite Parts is listed below, and our collection can be divided into three subcollections as basic parts, parts of the project pathway and parts for experimental verification.
We wish our design can provide inspiration to future protein-based detection system, and our experiment strategies can be furtherly developed to simplify protein-system projects. We hope that the parts and part collection can be further explored and improved, contributing to the overall community of synthetic biology.
Basic Parts
Our basic parts contain coiled-coil domains, fragments of the COI1-JAZ1 pathway, novel and improved proteases and reporter proteins. These are the fundamental sequence of proteins we used in our system.
Six coiled-coil domains composed the main section for our new basic parts. P3/AP4 and WinZip A2/B1 can form a heterodimer, while P3mS and AP4mS are affinity-reduced mutants.
JAZ1 degron peptide and COI1 are used in the second input, binding the phytotoxin COR. They could bind together and form a complex.
Proteases mainly consist of our new parts, AvrRpt2, and its cleavage site. We also uploaded the split PPV proteases and a catalytically inactive mutant of the cTEVp fragment.
Reporters are the section of the signal proteins containing trehalase, luciferase and GFP. We use these parts to produce output signals for us to detect.
| Section | Code | Name | Introduction |
|---|---|---|---|
| Coiled-coil domains | BBa_25JDZ4J4 | P3 Coiled-coil Domain | P3 is an artificial coiled-coil domain. It has a high binding affinity for AP4, and will preferentially form a P3/AP4 heterodimer. |
| Coiled-coil domains | BBa_25XSPMG1 | AP4 Coiled-coil Domain | AP4 is an artificial coiled-coil (CC) domain. It is the reverse peptide of another coiled-coil domain, P4, with higher binding affinity for P3. It will preferentially form a P3/AP4 heterodimer. |
| Coiled-coil domains | BBa_253ZVCBQ | WinZip A2 Coiled-coil Domain | WinZipA2 is an artificial coiled-coil domain. It has high binding affinity for WinZipB1, and will preferentially form a WinZipA2/B1 heterodimer. |
| Coiled-coil domains | BBa_25V40N0N | WinZip B1 Coiled-coil Domain | WinZipB1 is an artificial coiled-coil domain. It has high binding affinity for WinZipA2, and will preferentially form a WinZipB1/A2 heterodimer. |
| Coiled-coil domains | BBa_25O0NZS0 | P3mS Coiled-coil Domain | P3mS is an artificial coiled-coil domain derived from P3. It has lower structural stability and destabilizes the heterodimer formed with AP4. Therefore, P3mS can be replaced by P3 in the P3mS/AP4 heterodimer. |
| Coiled-coil domains | BBa_25V6OAZ3 | AP4mS Coiled-coil Domain | AP4mS is an artificial coiled-coil domain derived from AP4. It has lower structural stability and destabilizes the heterodimer formed with P3. Therefore, AP4mS can be replaced by AP4 in the P3/AP4mS heterodimer. |
| COI1-JAZ1 heterodimer | BBa_25OF4Z6A | JAZ1 degron peptide | JAZ1 degron peptide is a 20-aa peptide that derives from JAZ1 protein. It is the minimal sequence in JAZ1 required for high-affinity ligand binding with COI1 and sufficient for coronatine-induced COI1-JAZ1 interaction. |
| COI1-JAZ1 heterodimer | BBa_K1347003 | COI1 | COI1 exhibits strong interactions with JAZ1 degron peptide, leading to a high-affinity co-receptor to ligands, including JA or COR |
| Proteases | BBa_25MMLCX8 | AvrRpt2, Cysteine Protease Avirulence Protein | AvrRpt2 is a cysteine protease, which can specifically recognize a consensus cleavage site VPxFGxW (x represents an uncertain amino acid) |
| Proteases | BBa_25MVPE7S | AvrRpt2 Cleavage Site | The specific cleavage site of protease AvrRpt2. AvrRpt2 can specifically cleave at the consensus sequence VPxFGxW on the peptide, between the G and x residues. |
| Proteases | BBa_251TPK43 | nPPVp, codon-optimized for E.coli | nPPVp is the N-terminal fragment of plum pox virus protease (PPVp). Recombining with cPPVp, a functional PPV protease can be reassembled and regain its cleavage activity. |
| Proteases | BBa_259UGXOY | cPPVp, codon-optimized for E.coli | cPPVp is the C-terminal fragment of plum pox virus protease (PPVp). Recombining with nPPVp, a functional PPV protease can be reassembled and regain its cleavage activity. |
| Proteases | BBa_K2549014 | TEV protease N-terminal fragment | nTEVp is the N-terminal fragment of tobacco etch virus protease. Recombining with cTEVp, a functional TEV protease can be reassembled and regain its cleavage activity. |
| Proteases | BBa_K2549015 | TEV protease C-terminal fragment | cTEVp is the N-terminal fragment of tobacco etch virus protease. Recombining with nTEVp, a functional TEV protease can be reassembled and regain its cleavage activity. |
| Proteases | BBa_25JMGXPM | cTEVp*, catalytically inactive mutant of cTEVp | cTEVp* is the catalytically inactive mutant of cTEVp, which can be used as an autoinhibitor of nTEVp, blocking nTEVp and cTEVp recombination to prevent its cleavage ability. |
| Reporters | BBa_25IE55BV | Trehalase | Trehalase is a glycolytic enzyme that catalyzes the hydrolysis of trehalose into two glucose molecules. |
| Reporters | BBa_254TDHBX | Trehalase N-terminal Fragment (nTreA) | nTreA is the N-terminal fragment of trehalase. Recombining with cTreA, a functional trehalase can be reassembled and regain its catalytic function and ability to produce glucose. |
| Reporters | BBa_25LLF7T4 | Trehalase C-terminal Fragment (cTreA) | cTreA is the C-terminal fragment of trehalase. Recombining with nTreA, a functional trehalase can be reassembled and regain its catalytic function and ability to produce glucose. |
| Reporters | BBa_25KXO645 | Luciferase N-terminal Fragment (nLuc), codon-optimized for E. coli. | nLuc is the N-terminal fragment of firefly luciferase. Recombining with cLuc, a functional luciferase can be reassembled and regain its ability to emit luminescence. |
| Reporters | BBa_25T6SZQV | Luciferase C-terminal Fragment (cLuc), codon-optimized for E. coli. | cLuc is the C-terminal fragment of firefly luciferase. Recombining with nLuc, a functional luciferase can be reassembled and regain its ability to emit luminescence. |
| Reporters | BBa_K4759009 | GFP1-9 | GFP1-9 is the 1st - 9th β-fold fragments of GFP. Recombining with GFP10 and GFP11, a functional GFP can be reassembled and regain its ability to emit fluorescence. |
| Reporters | BBa_K4759010 | GFP10 | GFP10 is the 10th β-fold fragment of GFP. Recombining with GFP1-9 and GFP11, a functional GFP can be reassembled and regain its ability to emit fluorescence. |
| Reporters | BBa_K4759011 | GFP11 | GFP11 is the 11th β-fold fragment of GFP. Recombining with GFP1-9 and GFP10, a functional GFP can be reassembled and regain its ability to emit fluorescence. |
| Other | BBa_K3037001 | Maltose Binding Protein (MBP-tag) | Maltose binding protein is a well-established and reliable protein tag that can increase the solubility of proteins to which it is fused. |
| Other | BBa_K2406020 | T7-LacO Promoter | A promoter activated by T7 polymerase. It has a downstream Lac Operator, which is bound by LacI and repressed. |
| Other | BBa_K1088018 | LacI repressor from E. coli | The coding region for the LacI protein, which binds to the lac promoter and inhibits transcription. IPTG could bind LacI and inhibit its function. |
| Other | BBa_K2572009 | LacI promoter | A promoter that activates the expression of the LacI repressor. |
| Other | BBa_K731721 | T7 terminator | A short terminator by T7 phage. |
| Other | BBa_K914003 | L-rhamnose-inducible promoter (pRha) | A promoter that is capable of high-level recombinant protein expression in the presence of L-rhamnose. |
| Other | BBa_B0034 | RBS | A ribosome binding site. |
| Other | BBa_B0015 | double terminator (B0010-B0012) | A two-way terminator. |
| Other | Bba_J23119 | J23119 constitutive promoter | A strong constitutive promoter. |
Composite Parts
Parts of the project pathway
This is the section of composite parts of our project design. All of these parts can refer to the corresponding design in the pathway figure above.
| Section | Code | Name | Introduction |
|---|---|---|---|
| Input 1 | BBa_2518A672 | cTEVp-WinZipA2 | This is the coding sequence of fusion protein cTEVp-WinZipA2, combining coiled-coil domain WinZip A2 and C-terminal split TEV protease. |
| Input 1 | BBa_25LMVZ4Y | cTEVp*-WinZipB1-AvrRpt2s-TEVs-WinZipB1-nTEVp | This is the coding sequence of fusion protein cTEVp-WinZipA2, combining the coiled-coil domain WinZip A2 and the C-terminal split TEV protease. |
| Input 2 | BBa_253C22TU | COI1-nPPVp | This is the coding sequence of fusion protein COI1-nPPVp, combining COI1 and the N-terminal split PPV protease. |
| Input 2 | BBa_259RD21C | JAZ1 (reversed)-cPPVp | This is the coding sequence of fusion protein JAZ1 (reversed)-cPPVp, combining reversed JAZ1 degron peptide and the C-terminal split PPV protease. |
| Output | BBa_25BSL5PJ | AP4mS-TEVs-P3-GFP11 | This is the coding sequence of fusion protein AP4mS-TEV protease cleavage site-P3-GFP11. |
| Output | BBa_250RLQLX | AP4mS-TEVs-P3-cTreA | This is the coding sequence of fusion protein AP4mS-TEV protease cleavage site-P3-cTreA. |
| Output | BBa_25IYZ0KQ | AP4mS-TEVs-P3-cLuc | This is the coding sequence of fusion protein AP4mS-TEV protease cleavage site-P3-cLuc. |
| Output | BBa_25RRX9IJ | GFP10-AP4-PPVs-P3mS | This is the coding sequence of fusion protein GFP10-AP4-PPV protease cleavage sequence-P3mS. |
| Output | BBa_251UQNF9 | nTreA-AP4-PPVs-P3mS | This is the coding sequence of fusion protein nTreA-AP4-PPV protease cleavage sequence-P3mS. |
| Output | BBa_256YEGU6 | nLuc-AP4-PPVs-P3mS | This is the coding sequence of fusion protein nLuc-AP4-PPV protease cleavage sequence-P3mS. |
Parts for experimental verification
This is the section of composite parts designed for experimental verification. We designed several parts to verify the combination and rearrangement of coiled-coil domains, the output signals, COI1-JAZ1 interaction, cleavage activity and positive feedback.
| Section | Code | Name | Introduction |
|---|---|---|---|
| Coiled-coil domain verification | BBa_25DSNIG7 | nLuc-AP4 | This is the verification plasmid designed and constructed to verify the function of AP4 coiled-coil domain. |
| Coiled-coil domain verification | BBa_2527MQP2 | P3-cLuc | This is the verification plasmid designed and constructed to verify the function of P3 coiled-coil domain. |
| Output verification | BBa_25MD5KSC | MBP-nTreA-AP4 | This is the verification plasmid designed and constructed to verify the function of N-terminal trehalase (nTreA). |
| Output verification | BBa_25V4VNKA | P3-cTreA | This is the verification plasmid designed and constructed to verify the function of C-terminal trehalase (cTreA). |
| Output verification | BBa_258WFFPJ | Full-TreA plasmid | This is the verification plasmid designed and constructed to express and verify the construction and function of trehalase (TreA). |
| Output verification | BBa_25T299QB | Plasmid GFP1-9 | This is the verification plasmid designed and constructed to express and verify the function of beta-strand 1-9 of the split Green Fluorescent Protein. |
| Output verification | BBa_25EVV9VN | Plasmid GFP_Final | This is the verification plasmid designed and constructed to express and verify the function of beta-strands 10 and 11 of the split Green Fluorescent Protein. |
| Cleavage and rearrangement verification | BBa_257IGJVK | Plasmid A'-B-A-GFP | This is the verification plasmid designed and constructed to express and verify the function of the cleavage sites between CC-domains, and the rearrangement ability of CC-domains, expressing GFP10_WinZip B1_TEVs_WinZip B1, WinZip A2_GFP11, and GFP1-9. |
| Cleavage and rearrangement verification | BBa_25WZS0QB | Plasmid C-D-E-C'-GFP | This is the verification plasmid designed and constructed to express and verify the function of the cleavage sites between CC-domains, and the rearrangement ability of CC-domains, expressing GFP10_AP4_TEVs_P3mS, AP4mS_TEVs_P3_GFP11, and GFP1-9. |
| COI1-JAZ1 verification | BBa_25FZQVQU | MBP-COI1-GFP11 | This is the verification plasmid designed and constructed to express and verify the function of COI1. |
| COI1-JAZ1 verification | BBa_254RZXUU | MBP-GFP10-JAZ1 | This is the verification plasmid designed and constructed to express and verify the function of JAZ1 degron peptide. |
| MBP-tagged cTEVp | BBa_258NBWZH | MBP-cTEVp-WinZipA2 | This is the verification plasmid designed and constructed to fuse MBP tag with cTEVp-WinZipA2. |
| AvrRpt2 cleavage efficiency selection | BBa_25RPF424 | nLuc-AvrRpt2s-AP4 | This is the plasmid designed and constructed to select the cleavage site of AvrRpt2 that has the best efficiency. |
| Positive feedback verification | BBa_25LOPU94 | Plasmid nonTEV | This is the verification plasmid designed to verify the positive feedback pathway. |
