Difference between revisions of "Team:ETH Zurich/Basic Part"
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Revision as of 23:06, 30 October 2017
Basic Parts
Overview
All parts were codon-optimized for expression in E. coli Nissle with Geneious and modified to remove forbidden restriction sites.
| 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 |
Design
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 the absence of heme results in stronger iron retention within the protein scaffold while not decreasing the 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 and assembles into pores spanning the bacterial cell membrane which quickly results in 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: FIXME FIXME FIXME RBS info
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: 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 transcription. In design A, the distances between the intercalated promoter and the binding sites were taken from BBa_K1847007.
Organism: FIXME FIXME FIXME
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: FIXME FIXME FIXME
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: FIXME FIXME FIXME
Based on: BBa_K1847007 (O1 and O2) and BBa_R0062 (pLux)
Source: gBlock
Registry: BBa_K2500012
