Difference between revisions of "Team:ETH Zurich/Circuit"
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| + | <p><em>This page will tell you all about the design of the genetic circuit that makes CATE work. To read about how we developed the idea of CATE, visit the <a href="https://2017.igem.org/Team:ETH_Zurich/Description">Story of CATE.</a> To skip to story and jump directly to how CATE is designed to treat tumors, see CATE in Action.<a href="https://2017.igem.org/Team:ETH_Zurich/Applied_Design"> Circuit page.</a></em></p> | ||
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Revision as of 17:56, 31 October 2017
Circuit
This page will tell you all about the design of the genetic circuit that makes CATE work. To read about how we developed the idea of CATE, visit the Story of CATE. To skip to story and jump directly to how CATE is designed to treat tumors, see CATE in Action. Circuit page.
Overview
Genetic circuits and biological information processing are crucial elements of synthetic biology. For CATE we specifically designed a circuit able to integrate signals from both the outside and the inside of the body. It features two safety checkpoints to ensure superiority in terms of off-target damage and controllability compared to conventional cancer treatment strategies.
The genetic parts are located on two plasmids, the actuator plasmid and the regulator plasmid. On the regulator plasmid, genes used for control of the behavior are located. These include luxR, lldP and lldR designed to function as an AND gate (Tumor Sensor) and tlpA (Heat Sensor). Being combined in an operon on a single plasmid, it makes it easy to manipulate coding regions and fine-tune the gene expression level. The actuator plasmid contains genes for the actions CATE can take. The genes for bacterioferritin (MRI Contrast Agent), azurin (Anti-Cancer Toxin) and protein E (Cell Lysis) are located on this plasmid. Additionally, to enable quorum sensing (part of Tumor Sensor), the gene for LuxI is also on the actuator plasmid. In general, the regulator plasmid contains the sensors to integrate inputs from the outside world, whereas the actuator plasmid produces molecules that act on the environment.
FIXME with hover over
The Circuit
Checkpoint 1
Cate can sense tumors with a built-in tumor sensor. This is the first checkpoint of activation of the killing ability. The tumor sensor can sense the presence and abundancy of two specific molecules only available when CATE colonized a tumor. The tumor sensor consists of an artifical AND-gate promotor that activates downstream genes if lactate and AHL are present.
Tumor Sensor (AND gate promotor)
The term AND-gate stands for a logic gate, able to distinguish states of inputs and modulating the output according to the logic table of an and gate. The AND-gate integrates the signal of two binary inputs and gives an output signal if both inputs are available. Our synthetic AND-gate promotor works by looping the DNA using two operator regions flanking the LuxR binding site. LuxR is needed in a AHL bound dimeric state to enable transcription of the gene by the RNA Polymerase. LldR mediates the DNA looping by binding the two operator regions. It gets released from the DNA upon a conformational change initiated by binding of Lactate. If Lactate is present, LldR therefore gets released from the DNA and LuxR could bind to the promotor and initiate transcription. But if no AHL is present, LuxR does not initiate transcription and the promotor remains in the OFF-state. If AHL is present but no Lactate, the promotor also stays OFF, because LldR still loops the DNA and disables binding of LuxR-AHL.
See how the AND-gate works
Quorum Sensing
Quorum Sensinsing delivers the first input signal to the synthetic AND-gate promotor. It is derived from the bacterial ability to sense a the cell density in a biofilm or in the culture medium. It is used for biofilm formation, initiation of bioluminescence. It is used by bacteria in general to coordinate processes which would be inefficient if only a single bacterium does it - exactly what is needed for the production of the Anti-Cancer Agent of CATE. The system involves two proteins, LuxI and LuxR and the promotor region PLux. LuxI is an enzyme that produces AHL at a low but constant rate. AHL is an amphiphilic chemical compound that can easily travel through cell membranes. It binds to the own LuxR and to the one of other bacteria in the vicinity. Because LuxI expression is regulated by the PLux promotor region, a positive feedback appears: the more AHL from other bacteria binds to LuxR, the more can LuxR activate PLux and expression of LuxI, which then produces more AHL again.
But this positive feedback only gets enabled in CATE if Lactate is present and relieves the DNA looping.
Lactate Sensing
Lactate can be used as a growth substrate by bacteria. But they need to produce a specific set of enzymes to process lactate. Producing all these enzymes would be a waste of energy and is therefore highly controlled. Lactate Sensing is used to detect presence of lactate, and control the production of the protein needed for using Lactate as energy source. Lactate Sensing involves two proteins: LldR and LldP. While LldR in the absence of lactate dimerizes and forms a DNA loop, repressing transcription, LldP is needed for the influx of lactate through the cell wall into the cytoplasm.
MRI Contrast Agent
If the AND-gate promotor detects presence of both tumor related signals, it activates expression of the MRI Contrast Agent and the Anti Cancer Toxin. Bacterioferritin is one of the three forms of ferritin-like proteins found in bacteria. It has been shown that overexpression of bacterioferritin in E. coli Nissle can lead to a visible contrast change in MRI, which allows for visualization of the bacteria.
Anti-Cancer Toxin
Azurin is a type I blue copper protein with a molecular mass of 14 kDa originating from the well-known human pathogen Pseudomonas aeruginosa. It has already been successfully expressed in E. coli Nissle and used as an anti-cancer agent in vivo.
Checkpoint 2
Heat Sensor (TlpA repressor system)
The TlpA thermosensitive repression system is derived from Salmonella and belongs to the protein thermosensors. It consists of a constitutively expressed regulator protein called TlpA and an inducible operator-promotor called PTlpA. TlpA contains an approximately 300-residue coiled-coil domain at the C-terminus that uncoils between 42 °C and 45 °C. In low temperatures, its N-terminal domain is in a dimeric state and can bind the 52-bp PTlpA. Transcription of the downstream gene can therefore happen at temperatures above 42 °C but not below. [1][2]
Cell Lysis (Protein E)
Protein E is a protein produced by phage Phi X 147, which (in nature) lyses the host cell after production of phage particles. [2] The exact mechanism of action of protein E has long been controversial and different models were proposed to explain its lytic function. It has been suggested that protein E activates a component of the E. coli autolytic system, that it inhibits cell wall synthesis in a manner similar to penicillin or that it oligomerizes to form a transmembrane tunnel, all leading to release of cytoplasmic content and ultimately cell death. Independant of the exact mechanism, phage particles should be released to the extracellular space in the same way as our anti-cancer toxin should be released.
