The idea

From wiki iGEM Valencia

Valencia iGEM 2006 project

Our project for this iGEM edition in 2006 is make different modules in order to assemble it. Like that, we construct different devices. We distinct in two types of devices: sensors and actuators.

We are interested in sensing an interesting variable, such as a concentration of a nice molecule (vanillin), pH or light. So we construct this sensors using the appropiate E. coli strains. As actuator we use a genetic synthetic circuit that at high input levels has a given fluorescent response and at low levels other. Therefore, for intermediate levels there is a color gradient. We focuse our project designing a synthetic membrane receptor of vanillin.


Schematic view of our cellular biosensor based on E.Coli. The vanillin molecule crosses the external membrane, then it binds our synthetic vanillin-binding protein that changes its conformation to a closed form. This in turn binds the trg domain of a chimeric protein that transduces the signal to the EnvZ domain. The Ompr-P activates the transcription of the promoters of our actuator device.
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Schematic view of our cellular biosensor based on E.Coli. The vanillin molecule crosses the external membrane, then it binds our synthetic vanillin-binding protein that changes its conformation to a closed form. This in turn binds the trg domain of a chimeric protein that transduces the signal to the EnvZ domain. The Ompr-P activates the transcription of the promoters of our actuator device.


1. Sensor


The sensor is a system to produce a signal when it detects an input. We can work with many inputs (e.g., light, a molecule, pH, etc.). However the output is always the same: a phosphore that phosphorilates OmpR to give OmpR-P. Like that, we get a modularity if the receptor has OmpR-P as input.

Sensor module. We use OmpR-P as output (in fact, only P that phosphoriles the OmpR protein).
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Sensor module. We use OmpR-P as output (in fact, only P that phosphoriles the OmpR protein).


  • Molecule Sensor: Inspired on Hellinga’s work sensing TNT and other molecules

using a mutated periplasmic binding protein (PBP), our team thought in build a PBP that docks a vanillin molecule. It performs an allosteric motion that makes it binding to the trg protein. When the PBP-vanillin complex binds trg, then an allosteric motion is propagated to the EnvZ kinase domain resulting in autophosphorylation and phosphate transfer to OmpR transcription factor (OmpR-P).

  • pH sensor: using a EnvZ- E. coli strain and introducing into the cell a EnvZ CDS, we can use a promoter which is regulated (activated) by OmpR-P (PompC). Thus, putting a GFP CDS downstream the promoter, we can obtain a transfer function between GFP (for example) and pH. We think also in a cell-cell communication designing a cell be able to change the extracellular pH using a metabolic pathway producing, for instance, lactate or acetate acid.
  • Light sensor (UT Austin) is another alternative to use as a sensor to our genetic synthetic network.


2. Actuator


In this case, the actuator is a genetic network. It takes as input OmpR-P, and the output is whatever we want, for example a fluorescent protein (e.g., GFP).

If we use the vanillin sensor (and if it works), we will have designed an E. coli able to taste flavors (ECOLITASTER). On the other hand, we can assemble this circuit with the pH sensor or with the light sensor.

  • If we use the vanillin sensor (and if it works), we have E. coli tasting flavors: Power Point presentation. On the other hand, we can assemble this circuit with the pH sensor or with the light sensor.
Actuator module (genetic network). The transcription factor OmpR-P is charged to switch on this element.
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Actuator module (genetic network). The transcription factor OmpR-P is charged to switch on this element.

Mechanism:

  • OmpR-P regulates our three ompC-based promoters. There are two possibilities to make a repressed by OmpR-P promoter: first, putting the operator downstream the promoter; second: using an intermediate inverter. We think that it is easier to use an inverter.
  • The threshold concentration of OmpR-P that activates the promoter of the CRP gene should be lower than the one repressing the promoter of the cI gene (or lower than the effective threshold concentration using an intermediate inverter). Therefore, at intermediate concentrations of OmpR-P, CRP and cI are expressed.
  • Thus the AND promoter is activated and GFP is expressed at intermediate concentrations of OmpR-P.
From Bintu et al., Transcriptional regulation by the numbers: applications. Current Opinion in Genetics & Development 2005, 15:125–135
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From Bintu et al., Transcriptional regulation by the numbers: applications. Current Opinion in Genetics & Development 2005, 15:125–135
  • The cells’ color is: At high vanillin concentration: red. At intermediate: yellow. At low: green. No vanillin: no color. If our response does not allow to have an AND behavior, but a NAND, we have red at high vanillin concentration, yellow at intermediate, and green at low and nothing input.


List of parts:

  • A CDS corresponding to a fusion protein between a designed periplasmic binding protein (PBP) docking vanillin and a transmembrane histidine kinase (Looger et al. Nature 2003).
Computational design of PBP domain. Make corresponding BioBrick.
  • A promoter with two regulators (activators) using AND logic (Joung et al. Science 1994).
Part already designed. Make BioBrick.
  • Two promoters activated by OmpR-P (i.e. ompC promoter).
The first is already a part in the Registry.
  • Design a mutation of OmpR-P operator to reduce binding.
Promoter repressed by OmpR-P.
  • Graft OmpR-P operator downstream of a promoter. Mutate operator to reduce binding if necessary. Make BioBrick.
  • Four CDS:
Coding cI part from the Registry.
Coding CRP from Joung et al. Make BioBrick.
Two reporters: GFP & RFP, they should have similar expression levels.
Change codon usage in CDS to adjust their expression levels. Make BioBrick.


Alternative genetic network

We have an alternative plan if the first fails. So far, we have considered a circuit with a promoter that is twice positively regulated with an AND logic. This AND gate, we have to construct it using primers. We have taken the sequence using a free software on Internet we have built these primers that anneal between them in order to give the complete DNA sequence.

Now we consider another possibility. We use only unitary promoters, namely, only one protein can regulate it. We also use well-known proteins, in fact, the same ones. In addition, we can see the parts of the circuit, but also the devices. This design takes advantege of the constructions (composite parts) from the wells that MIT sent.

One important thing is that, in this circuit, OmpR promoters (with different binding to activation) are changed. And the internal inverter (cI inverter) has to be strong, namely, the basal rate of tetR downstream the promoter by cI regulated (commonly called PR) has to be approximately zero, in order to get a well behavior.

Alternative circuit using an internal inverter. The bahavior, a priori, is the same. In this circuit there are 8 devices and it requires 8 standard assemblies.
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Alternative circuit using an internal inverter. The bahavior, a priori, is the same. In this circuit there are 8 devices and it requires 8 standard assemblies.

3. Conclusions:

  • The actuator is a simple circuit with only 4 or 5 genes.
  • We use well known proteins: cI, CRP, GFP, RFP.
  • It allows to design a cool “taste” sensor (e.g. a protein that senses vanillin). E. Coli that tastes! Also a pH sensor.
  • We use a standard two-component signal transduction system part and we could use other sensors (e.g. UT Austin’s light sensor or Hellinga’s TNT sensor) if ours fails.
  • It is a robust system.
  • It allows to add new parts (e.g. a promoter with AND logic) to the Registry and design new ones (e.g. a promoter repressed by OmpR-P).
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