Team:McMaster/Parts
Parts
Parts Overview
| BASIC PARTS (n=17) | ||||
|---|---|---|---|---|
| Part Name & Number | Type | Description | Designer (iGEM Group) | Length (bp) |
| Constitutive Promoter (BBa_J23100) | Promoter | Well-characterized constitutive promoter that will allow downstream sequences to be expressed at all times. | John Anderson (iGEM2006_Berkeley) | 35 |
| PyeaR Promoter (BBa_K216005) | Promoter | Promoter native in the Escherichia coli yeaR/yoaG operon, sensitive to nitric oxide, nitrate, and nitrite. | Edinburgh iGEM 2009 | 135 |
| flhDC promoter BBa_K554001 | Promoter | Promoter native to Escherichia coli that initiates transcription in response to phosphorylated-QseB. | UNICAMP EMSE Brazil team (iGEM11_Brazil-France) | 85 |
| Ribosome Binding Site BBa_B0034 | RBS | Well-characterized strong RBS from the Community collection to efficiently express colicin proteins. | Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr (Antiquity) | 12 |
| GSG linker+T2A (BBa_K1537017) | Coding | Cleavage linker with improved ability to separate two protein products expressed under the same promoter. | Jie Li (iGEM14_UESTC-GreenLife) | 63 |
| QseC Histidine Kinase Sensor (BBa_M36234) | Coding | Codes for QseC histidine kinase sensor that auto-phosphorylates in the presence of autoinducer-3 and catecholamines and phosphorylates QseB to regulate gene expression. | Lawrence Xing (Stanford BIOE44 - S11) | 1350 |
| QseB Regulatory Protein (BBa_M36231) | Coding | Codes for QseB response regulator protein that is phosphosylated by QseC in the presence of autoinducer-3 and catecholamines. Can bind to flhDC promoter to regulate downstream gene expression. | Lawrence Xing (Stanford BIOE44 - S11) | 660 |
| NarL Regulatory Protein (BBa_K3411040) | Coding | Codes for response regulator protein that is phosphorylated by NarX in the presence of nitrate. Can bind to PyeaR promoter to regulate downstream gene expression. | Janet Standeven, Nikhita Lalwani, Sindhura Mettupalli, Kathy Ye, Sachintha Ashok, Saif Khan (iGEM20_Lambert_GA) | 651 |
| TorA Signal Sequence BBa_K3114005 | Coding | Signaling peptide that allows for secretion of proteins via the Tat secretion pathway. | Cassandra Sillner, Sara Far, Sravya Kakumanu, Nimaya De Silva, Andrew Symes (iGEM19_Calgary) | 138 |
| Colicin E9 ImmunityProtein (BBa_K1962005) | Coding | Protein dimerizes with colicin E9 to prevent bacteriocin activity within the host bacteria. | Frank Sargent (iGEM16_Dundee) | 258 |
| 2A Linker Sequence (BBa_K2976010) | Conjugation | Linker allows for efficient transcription of the proteins in the second ORF and cleavage between two proteins. | Jiatong Chen (iGEM19_CPU_CHINA) | 63 |
| Terminator (BBa_B0010) | Terminator | Well-characterized terminator to end gene expression of a circuit. | Randy Rettberg (Antiquity) | 80 |
| Double Terminator (BBa_B0014) | Terminator | Well-characterized terminator to end gene expression of a circuit. | Reshma Shetty (Antiquity) | 95 |
| pSEVA511 (GENBANK #JX560341) | Backbone | Tetracycline (Tc) resistant plasmid backbone vector from the pSEVA collection with restriction sites (PacI, SPel) flanking the cargo region. | Victor De Lorenzo | 2330 |
| pSEVA311 (GENBANK #JX560331) | Backbone | Chloramphenicol (Cm) resistant plasmid backbone vector from the pSEVA collection with restriction sites (PacI, SPel) flanking the cargo region. | Victor De Lorenzo | 1838 |
| pSEVA611 (GENBANK #JX560346) | Backbone | Gent(Gm) resistant plasmid backbone vector from the pSEVA collection with restriction sites (PacI, SPel) flanking the cargo region. | Victor De Lorenzo | 1871 |
| pSEVA211 (GENBANK# JX560326) | Backbone | Kanamycin (Km) resistant plasmid backbone vector from the pSEVA collection with restriction sites (PacI, SPel) flanking the cargo region. | Victor De Lorenzo | 1993 |
Table of New Parts by Detection Module
| NEW BASIC PARTS (n=4) | ||||
|---|---|---|---|---|
| Part Name & Number | Type | Description | Source | Length (bp) |
| Isolated Colicin E1 with 10x His-tag (BBa_K4010001) | Coding | Bacteriocin, kills Escherichia coli via pore-formation. Attached to histidine-tag for purification purposes | IGem registry parts BBa_K822002, BBa_K844000 | 1602 |
| Isolated Colicin E1 Immunity Protein (BBa_K4010002) | Coding | Immunity protein preventing colicin E1 from killing the host bacteria. | IGem registry parts BBa_K822002 | 342 |
| Colicin E9 with His-tag and RBS (BBa_K4010003) | Coding | Bacteriocin, kills Escherichia coli via DNase activity. Attached to histidine-tag for purification purposes. | IGem registry parts BBa_K242201, BBa_K844000 | 457 |
| NarX Histidine Kinase Sensor (BBa_K4010006) | Coding | Histidine kinase sensor, auto-phosphorylates in response to nitrate and phosphorylates NarL to regulate gene expression. | NCBI Gene ID: 945788 | 1797 |
| NEW COMPOSITE PARTS (n=8) | ||||
| TorA-Linked Colicin E1 and Immunity Protein (BBa_K4010004) | Composite | His-tagged colicin E1 able to be secreted without lysis via TAT export pathway linked to an immunity protein to prevent killing the host-cell. | TorA sequence (BBa_K3114005) Colicin E1 (BBa_K4010001) 2A-Linker (BBa_K2976010) Colicin E1 Immunity protein (BBa_K4010002) | 2169 |
| TorA-Linked Colicin E9 and Immunity Protein (BBa_K4010005) | Composite | His-tagged colicin E9 able to be secreted without lysis via TAT export pathway linked to an immunity protein to prevent killing the host-cell. | TorA sequence (BBa_K3114005) Colicin E9 (BBa_K4010003) 2A-Linker (BBa_K2976010) Colicin E9 ImmunityProtein (BBa_K1962005) | 936 |
| Pconst + QseC + NarX & PyeaR + QseB + NarL (BBa_K4010007) | Composite | AND Gate sensor of autoinducer-3 and nitric oxide; includes positive feedback system to amplify NarL expression after nitric oxide undergoes oxidation into nitrate. Can be paired with another effect circuit under the flhDC promoter. *NOTE*: This part is similar to BBa_K4010010 but has QseC and NarX connected with a linker instead of being under separate constitutive promoters. | Histidine Kinase Module: Pcon (BBa_J23100) RBS (BBa_B0034) QseC (BBa_M36234) Linker (BBa_K1537017) NarX (BBa_K4010006) Terminator (BBa_B0010) Regulator Module: PyeaR (BBa_K216005) RBS (BBa_B0034) QseB (BBa_M36231) Linker (BBa_K1537017) NarL (BBa_K3411040) Terminator (BBa_B0010) | 4983 |
| Pconst + QseC + NarX (BBa_K4010008) | Composite | Histidine kinase module of composite part BBa_K4010007; constitutively express QseC and NarX sensors with a cleavage linker between them. | Pcon (BBa_J23100) RBS (BBa_B0034) QseC (BBa_M36234) Linker (BBa_K1537017) NarX (BBa_K4010006) Terminator (BBa_B0010) | 3373 |
| PyeaR + QseB + NarL (BBa_K4010009) | Composite | Response regulator module of composite parts BBa_K4010007 and BBa_K4010010. Contains NarL which can be activated by NarX to amplify its own production along with QseB by binding to PyeaR. | PyeaR (BBa_K216005) RBS (BBa_B0034) QseB (BBa_M36231) Linker (BBa_K1537017) NarL (BBa_K3411040) Terminator (BBa_B0010) | 1602 |
| Pconst + QseC & Pconst + NarX & PyeaR + QseB + NarL (BBa_K4010010) | Composite | AND Gate sensor of autoinducer-3 and nitric oxide; includes positive feedback system to amplify NarL expression after nitric oxide undergoes oxidation into nitrate. Can be paired with another effect circuit under the flhDC promoter. *NOTE*: This part is similar to BBa_K4010007 but has QseC and NarX under separate constitutive promoters instead of being connected with a linker. This is to ensure that each module has less than 2000 bp and is ideal for DNA synthesis. | QseC Module: Pcon (BBa_J23100) RBS (BBa_B0034) QseC (BBa_M36234) Terminator (BBa_B0010) NarX Module: Pcon (BBa_J23100) RBS (BBa_B0034) NarX (BBa_K4010006) Terminator (BBa_B0010) Regulator Module: PyeaR (BBa_K216005) RBS (BBa_B0034) QseB (BBa_M36231) Linker (BBa_K1537017) NarL (BBa_K3411040) Terminator (BBa_B0010) | 5063 |
| Constitutively Expressed QseC (BBa_K4010011) | Composite | Constitutively expressed QseC histidine kinase sensor; can be paired with QseB in another circuit to activate downstream flhDC promoter. | Pcon (BBa_J23100) RBS (BBa_B0034) QseC (BBa_M36234) Terminator (BBa_B0010) | 1499 |
| Constitutively Expressed NarX (BBa_K4010012) | Composite | Constitutively expressed NarX histidine kinase sensor; can be paired with NarL in another circuit to activate downstream PyeaR promoter. | Pcon (BBa_J23100) RBS (BBa_B0034) NarX (BBa_K4010006) Terminator (BBa_B0010) | 1946 |
Composite Parts
TorA-Linked Colicin E1 and Immunity Protein BBa_K4010004
The colicins E1 (BBa_K4010001), modified from BBa_K822002, will be tagged with the TorA signal sequence at the N-terminal. The TorA signal (BBa_K3114005) is more effective than other tested signal sequences in transporting fully or partially folded proteins into the periplasm. Along with the 2A-like linker (BBa_K2976010) separating the colicin from the immunity protein (BBa_K4010002), this ensures the complete cleavage and permits E1 colicin to migrate through the inner membrane using the native Tat transportation system. Secretion into the extracellular space is carried out by the Type 2 secretion system into the native general secretory pathway. The colicin part also has a 10x his tag at the C-terminal for purification and quantification purposes.
TorA-Linked Colicin E9 and Immunity Protein BBa_K4010005
Similar to BBa_K4010004, part BBa_K4010005 is composed of the colicin E9 (BBa_K401002), modified from BBa_K242201, tagged with the TorA signal sequence (BBa_K3114005) at the N-terminal. The 2A-like linker (BBa_K2976010) separating the colicin from the immunity proteins (BBa_K1962005) allows the E9-E9 immunity heterodimer to use the Tat transportation system following secretion into the extracellular space by Type 2 secretion system. The colicin part has a 10x his-tag for affinity chromatography purification.
Pconst + QseC + NarX & PyeaR + QseB + NarL Composite Part (BBa_K4010007)
QseBC and NarXL are both two-component regulatory systems found in Escherichia coli to regulate gene expression (Zhou et al., 2003). Both QseC and NarX are histidine kinase sensors, responsive to autoinducer-3 (AI-3) and nitrate (NO3-) respectively. Upon activation, they both auto-phosphorylate and subsequently phosphorylate QseB and NarL regulatory proteins respectively, if they are present. These proteins are responsible for controlling downstream gene expressions: QseB can bind to flhDC promoter (BBa_K554001), while NarL can bind to PyeaR promoter (BBa_K216005).
This composite part can be used to activate genes downstream of a flhDC promoter (BBa_K554001) in the presence of both AI-3, a quorum sensing molecule, and nitric oxide, which has a short half-life and is oxidized into nitrate in the presence of oxygen and hemoglobin (Hakim et al., 1996). In E. coli, NsrR repressors are naturally present to inhibit PyeaR expression (Vine et al., 2011). When nitric oxide is present, NsrR will no longer bind to and repress PyeaR, allowing the QseB gene to be expressed (Lin et al., 2007). Once QseB is made, the system will react to AI-3 as sensed by QseC, and genes downstream flhDC promoter can be transcribed.
In our project, both nitric oxide and autoinducer-3 (AI-3) are used as biomarkers of adherent-invasive E. coli (AIEC) induced gut inflammation (Bretin et al., 2018). To prolong the effect of nitric oxide in our circuit, we proposed a positive feedback loop design using NarX/L’s ability to activate the yeaR-yoaG operon upon sensing nitrate. Specifically, the PyeaR promoter (BBa_K216005) in this operon contains a consensus sequence with the binding sites of the NsrR repressor (BBa_K1682011) and the NarL activator (BBa_K3411040) (Lin et al., 2007). In other words, the PyeaR promoter is simultaneously activated by NarL in the presence of nitrate (as sensed by NarX) and repressed by the NsrR protein in the absence of nitric oxide (Lin et al., 2007). As such, provided that NarX is constitutively expressed, NarL can be placed downstream of PyeaR along with a chosen sequence to ensure that oxidation of nitric oxide into nitrate would not pause downstream gene expression but instead amplify it.
References
- Hakim, T. S., Sugimori, K., Camporesi, E. M., & Anderson, G. (1996). Half-life of nitric oxide in aqueous solutions with and without haemoglobin. Physiological Measurement, 17(4), 267–277. https://doi.org/10.1088/0967-3334/17/4/004
- Lin, H.-Y., Bledsoe, P. J., & Stewart, V. (2007). Activation of yeaR-yoaG Operon Transcription by the Nitrate-Responsive Regulator NarL Is Independent of Oxygen- Responsive Regulator Fnr in Escherichia coli K-12. Journal of Bacteriology, 189(21), 7539–7548. https://doi.org/10.1128/JB.00953-07
- Noriega, C. E., Schmidt, R., Gray, M. J., Chen, L.-L., & Stewart, V. (2008). Autophosphorylation and Dephosphorylation by Soluble Forms of the Nitrate-Responsive Sensors NarX and NarQ from Escherichia coli K-12. Journal of Bacteriology, 190(11), 3869–3876. https://doi.org/10.1128/JB.00092-08
- Vine, C. E., Purewal, S. K., & Cole, J. A. (2011). NsrR-dependent method for detecting nitric oxide accumulation in the Escherichia coli cytoplasm and enzymes involved in NO production. FEMS Microbiology Letters, 325(2), 108–114. https://doi.org/10.1111/j.1574-6968.2011.02385.x
- Zhou, L., Lei, X.-H., Bochner, B. R., & Wanner, B. L. (2003). Phenotype MicroArray Analysis of Escherichia coli K-12 Mutants with Deletions of All Two-Component Systems. Journal of Bacteriology, 185(16), 4956–4972. https://doi.org/10.1128/JB.185.16.4956-4972.2003