Team:ETH Zurich/Basic Part
Basic Parts
You can read details about the design of our Basic Parts here. To check out all the parts that we created, visit Parts.
Overview
| Part | Description | BioBrick |
|---|---|---|
| Best Basic Part: AND Gate B |
Synthetic promoter responsive to LldR and luxR | BBa_K2500011 |
| Bacterioferritin | Heme-deletion mutant of bacterial iron storage protein functioning as an MRI contrast agent | BBa_K2500000 |
| Azurin | Redox protein originating from P. aeruginosa with cytotoxic activity | BBa_K2500001 |
| p28 | Effector domain of azurin | BBa_K2500002 |
| PTlpA | Temperature-responsive promoter optimized for slight activation above 37°C and full activation at 45 °C | BBa_K2500003 |
| TlpA | Temperature-dependent transcriptional repressor of pTlpa | BBa_K2500004 |
| RBS_TlpA | Temperature-dependent transcriptional repressor of pTlpa and synthetic RBS | BBa_K2500005 |
| Protein E | Bacteria-lysing protein encoded by phage Phi X 17 | BBa_K2500006 |
| RBSeng_TlpA | Temperature-dependent transcriptional repressor of pTlpa with engineered RBS | BBa_K2500007 |
| RBSeng_ProteinE | Bacteria-lysing protein encoded by phage Phi X 174 with engineered RBS | BBa_K2500009 |
| AND Gate A | Synthetic promoter responsive to LldR and luxR | BBa_K2500010 |
| AND Gate C | Synthetic promoter responsive to LldR and luxR | BBa_K2500012 |
| PConst_RBS_LldP/ LldR_PConst_RBS_LuxR |
Expression cassette consisting of LuxR and LldP/LldR | BBa_K2500013 |
Design
All parts were codon-optimized for expression in E. coli Nissle with Geneious and modified to remove forbidden restriction sites.
BBa_K2500000: Bacterioferritin
Design notes: Bacterioferritins are bacterial iron storage proteins. It has been shown that overexpression of bacterioferritin in E. coli Nissle 1917 can lead to a visible contrast change in MRI, which allows for visualization of the bacteria. We used a heme-deletion mutant as it does not decrease contrast change.
Organism: Escherichia coli Nissle
Based on: BBa_K1438001
Source: gBlock
Registry: BBa_K2500000
BBa_K2500001: Azurin
Design notes: Azurin is a redox protein with the ability to induce apoptosis in mammalian cells upon internalization. We removed the native signal peptide which is part of the pre-protein form of azurin as no targeting within the bacterial host cells is required.
Organism: Pseudomonas aeruginosa
Based on: BBa_K835004
Source: gBlock
Registry: BBa_K2500001
BBa_K2500002: p28
Design notes: The effector domain of azurin comprises amino acids 50 to 77. We extracted the sequence, introduced a start codon (ATG) at the beginning and two stop codons (TAA TAA) at the end. By encoding only the effector, we were aiming at achieving faster expression levels and a higher cytotoxic load carried by the bacteria.
Organism: Pseudomonas aeruginosa
Based on: BBa_K2500001
Source: gBlock
Registry: BBa_K2500002
BBa_K2500003: pTlpA
Design notes: The pTlpA promoter regulates the lysis step of CATE. The promoter is bound and inhibited by TlpA repressor proteins below 37 °C, thereby blocking the release of the cytotoxic compound at body temperature. To fully activate the promoter and thus cell lysis, the desired area is heated to 45 °C with focused ultrasound.
Organism: Salmonella typhimurium
Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.
Source: oligonucleotide sequence
Registry: BBa_K2500003
BBa_K2500004: TlpA
Design notes: TlpA is a constitutively expressed repressor protein which inhibits transcription of genes downstream of the TlpA promoter by binding to it. The binding to the promoter is reversibly deactivated by temperatures above 37 °C with full deactivation at 45 °C.
Organism: Salmonella typhimurium
Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.
Source: gBlock
Registry: BBa_K2500004
BBa_K2500005: RBS_TlpA
Design notes: Parameters influencing the final expression levels of the genes downstream of pTlpA include the RBS of its repressor protein TlpA. By tweaking these sequences, it should be possible to obtain a wide range of leakiness and fold changes (fold-change up to 200-fold).
Organism: Salmonella typhimurium
Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.
Source: gBlock
Registry: BBa_K2500005
BBa_K2500006: Protein E
Design notes: Protein E is a lysis protein originally encoded by the bacteriophage Phi X 147. It is now generally accepted that the most probable cellular target of protein E is the enzyme translocase I, encoded by the mraY gene. Translocase I is required for cell wall biosynthesis and its inhibition results to cell lysis.
Organism: Phage Phi X 174
Based on: provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich
Source: plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich
Registry: BBa_K2500006
BBa_K2500007: RBSeng_TlpA
Design notes: A RBS library was created using the Red Libs algorithm to find variants translating more TlpA RNA in order to create a thermoswitch tight enough to repress the premature induction of cell lysis by protein E.
Organism: Salmonella typhimurium
Based on: Piraner, Dan I., et al. Nature Chemical Biology 13.1 (2017): 75-80.
Source: FIXME RedLibs + gBlock
Registry: BBa_K2500007
BBa_K2500009: RBSeng_ProteinE
Design notes: A RBS library was created using the Red Libs algorithm to find variants translating less protein E RNA in order to prevent premature lysis of the bacterial cells.
Organism: Phage Phi X 174
Based on: provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich
Source: FIXME RedLibs + plasmid provided by Dr. Irene Wüthrich from Panke lab at the D-BSSE of ETH Zurich
Registry: BBa_K2500009
BBa_K2500010: AND Gate A
Design notes: The AND gate integrates two signals, namely the presence of high L-lactate concentrations and high bacterial cell density (qorum sensing) and regulates the effector functions of CATE. In the absence of high concentrations of L-lactate, LldR inhibitor proteins bind to the binding sites O1 and O2 surrounding the pLux promoter leading to the formation of a DNA loop. The pLux promoter is sequestered and inaccessible for transcriptional activation by the quorum sensing components. In design A, the distances between the intercalated promoter and the binding sites were taken from BBa_K1847007.
Organism: Escherichia coli, Vibrio fischeri
Based on: BBa_K1847007 (O1 and O2) and BBa_R0062 (pLux)
Source: gBlock
Registry: BBa_K2500010
BBa_K2500011: AND Gate B
Design notes: In design B, each binding site was duplicated in order to achieve a potential zipper mechanism and stronger inhibition due to binding more LldR inhibitor proteins.
Organism: Escherichia coli, Vibrio fischeri
Based on: BBa_K1847007 (O1 and O2) and BBa_R0062 (pLux)
Source: gBlock
Registry: BBa_K2500011
BBa_K2500012: AND Gate C
Design notes: In design C, an artificial spacer was embedded between the pLux promoter and the O2 binding site in order to influence the looping dynamics.
Organism: Escherichia coli, Vibrio fischeri
Based on: BBa_K1847007 (O1 and O2) and BBa_R0062 (pLux)
Source: gBlock
Registry: BBa_K2500012
BBa_K2500013: pConst1_RBS_LldP/LldR_pConst2_RBS_LuxR
Design notes:This expression cassette regulates the two inputs relayed to our AND gate, namely lactate and quorum sensing. LLdp is a L-lactate permease which transports lactate across the bacterial cell membrane. High lactate concentrations inhibit the LldR repressor proteins which under low-lactate conditions prevent access to the pLux quorum sensing promoter by LuxR-AHL complexes.
Higher bacterial cell densities result in a higher density of AHL communication molecules. AHL molecules diffuse across bacterial cell membranes, bind the transcription activator LuxR and activate the transcription of genes downstream of the pLux promoter, which is only accessible under high lactate levels as described above.
Organism: Escherichia coli, Vibrio fischeri
Based on: FIXME BBa_J23118 (pConst1), BBa_J23100 (pConst2), BBa_K1847007 (LldP/LldR) and BBa_C0062 (LuxR)
Source: FIXME
Registry: BBa_K2500013
