Team:UNAM-Genomics Mexico/Modules

From 2010.igem.org

(Difference between revisions)
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==Individual Modules==
==Individual Modules==
-
We decided to break down our device into 3 sub-devices: Reception, Emission, and Transmission. The rationale is as follows: the machinery that transforms the red input into chemical information is independent from the machinery that transforms chemical information into green output, and both are quite different from what transmits the information. Therefore, we can work with & model these three sub-devices.
+
We decided to break down our device into 3 sub-devices: Reception, Emission, and Transmission. The rationale is as follows: the machinery that transforms the red input into chemical information is independent from the machinery that transforms chemical information into green output, and both are quite different from what transmits the information. Therefore, we can work with & model these six sub-devices.
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We plan on constructing our reporter genes under the OmpF promoter and starting our system in a <Dark> state (where active OmpR concentration is high). Thus we hope to achieve an <IF Light> logic gate by using Cph8 as a sensing mechanism, and OmpF as a response one.
We plan on constructing our reporter genes under the OmpF promoter and starting our system in a <Dark> state (where active OmpR concentration is high). Thus we hope to achieve an <IF Light> logic gate by using Cph8 as a sensing mechanism, and OmpF as a response one.
-
 
-
The input for this sub-device is light, the output is Polymerases per Second.
 
See also, [http://partsregistry.org/Coliroid Coliroid].
See also, [http://partsregistry.org/Coliroid Coliroid].
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While the oxidation per se does not generate light, it does generate an intermediate molecule in an electronically exited state. When said molecule returns to a basal energy state, a photon is released.
While the oxidation per se does not generate light, it does generate an intermediate molecule in an electronically exited state. When said molecule returns to a basal energy state, a photon is released.
-
As you may imagine, these genes constitute an Operon. This is the Lux Operon from Vibrio fischeri. The input for this sub-device is Polymerases per Second, and the output is light.
+
As you may imagine, these genes constitute an Operon. This is the Lux Operon from Vibrio fischeri.
See also the work of [http://2009.igem.org/Team:Edinburgh/biology(biobricks) Edinburgh 2009].
See also the work of [http://2009.igem.org/Team:Edinburgh/biology(biobricks) Edinburgh 2009].
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When our regulator is in a phosphorilate state, it shows greater affinity for DNA. Thus it is active. The target promoter region has been recently identified. We thus plan on constructing our reporter genes under this promoter. Such a construction would be an <IF Light> logic gate. This system is quite similar to the EnvZ-OmpR system.
When our regulator is in a phosphorilate state, it shows greater affinity for DNA. Thus it is active. The target promoter region has been recently identified. We thus plan on constructing our reporter genes under this promoter. Such a construction would be an <IF Light> logic gate. This system is quite similar to the EnvZ-OmpR system.
-
 
-
The input for this sub-device is light, the output is Polymerases per Second.
 
See also [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474522/ this paper].
See also [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474522/ this paper].
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While the oxidation per se does not generate light, it does generate an intermediate molecule in an electronically exited state. When said molecule returns to a basal energy state, a photon is released. Likewise, LumP does not actually shift the spectrum, but the enzyme generates a substrate that does.
While the oxidation per se does not generate light, it does generate an intermediate molecule in an electronically exited state. When said molecule returns to a basal energy state, a photon is released. Likewise, LumP does not actually shift the spectrum, but the enzyme generates a substrate that does.
-
As you may imagine, these genes (sauf LumP) constitute an Operon. This is the Lux Operon from Vibrio fischeri. The input for this sub-device is Polymerases per Second, and the output is light.
+
As you may imagine, these genes (sauf LumP) constitute an Operon. This is the Lux Operon from Vibrio fischeri.
See also the work of [http://2009.igem.org/Team:Edinburgh/biology(biobricks) Edinburgh 2009].
See also the work of [http://2009.igem.org/Team:Edinburgh/biology(biobricks) Edinburgh 2009].
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 +
===Blue Reception===
 +
====Description====
 +
Blue Reception is composed of a slightly complicated system. Firstly, we have a quimeric sensing protein (LovTAP). This protein is composed of a sensing domain (a Light-Oxygen-Voltage domain) as well as the TAP protein from the Triptophan pathway. This protein will dimerize when struck by blue light. As a dimer, it shows greater affinity for DNA at the TrpO promoter.  However, it acts as an inhibitor to transcription. Occupied TrpO is repressed while free TrpO is active.
 +
 +
Our construction is consequently based on an "inhibit the inhibitor" logic. By constructing our reporter genes under a repressed promoter (TetO), and having LovTAP repress the inhibitor of said promoter (TetR), we establish a direct <IF Light> logic gate.
 +
 +
See also [http://partsregistry.org/Part:BBa_K191003 LovTAP].
 +
 +
 +
====Signaling Cascade====
 +
 +
When our device is struck by blue light, the following cascade will ensure:
 +
* Photon input
 +
* Lov-domain conformational change
 +
* LovTAP dimerization
 +
* TrpO promoter is repressed
 +
* Concentration of TetR collapses
 +
* TetO promoter is free
 +
* Pops output
 +
 +
 +
===Red Emission===
 +
 +
 +
====Description====
 +
 +
Red Emission is composed of mainly two enzymes. Our first enzyme (Luciferase) is a mutated form of the wild type enzyme found in Photinus pyralis. Our mutant is expected to glow red instead of the wild type blue-green. This enzyme catalyzes the oxidation reaction that yields light. The substrate for this reaction (Luciferin) is a most complicated molecule, and to our knowledge no one has ever managed to produce it within an E. coli chassis. Therefore, we need to inoculate the medium with luciferin to enable the reactions. However, recently a new enzyme was discovered (LRE) that recycles luciferin. We thus need only an initial inoculation with luciferin and from there on, the system is sufficiently autonomous.
 +
 +
We plan on using Luciferase as a reporter gene, while having LRE expressed constitutively.
 +
 +
For the Red Emission mutation, see [http://dx.doi.org/10.1016/j.ab.2005.07.015 this paper].
 +
 +
For the Red Emission protein, see [http://partsregistry.org/Part:BBa_I712019:Design this BioBrick part].
 +
 +
 +
====Signaling Cascade====
 +
 +
* Pops input
 +
* Luciferase downstream of TetO is transcribed
 +
* Oxidation of substrate
 +
* Photon output
}}
}}

Revision as of 21:12, 2 August 2010



Individual Modules

We decided to break down our device into 3 sub-devices: Reception, Emission, and Transmission. The rationale is as follows: the machinery that transforms the red input into chemical information is independent from the machinery that transforms chemical information into green output, and both are quite different from what transmits the information. Therefore, we can work with & model these six sub-devices.


Red Reception

Description

Red Reception is composed of a two-component system. Firstly, the chimeric protein Cph8 possess a light-sensing domain (PCB) and a histidine kinase domain (EnvZ). The chromophore has two states, and light triggers the passage of state. Thus, under dark conditions Cph8 shows kinase activity; under light conditions it does not.

Cph8's substrate is OmpR, a well studied Transcription Factor. When phosphorilated it shows greater affinity for DNA. OmpR regulates two promoters in an antagonistic way: in high concentrations of active OmpR, OmpC is active and OmpF is repressed. In low concentrations of active OmpR, OmpC is repressed and OmpF is active.

We plan on constructing our reporter genes under the OmpF promoter and starting our system in a <Dark> state (where active OmpR concentration is high). Thus we hope to achieve an <IF Light> logic gate by using Cph8 as a sensing mechanism, and OmpF as a response one.

See also, Coliroid.


Signaling Cascade

When our device is struck by red light, the following cascade will ensure:

  • Photon input
  • PCB conformation change
  • EnvZ kinase activity abolished
  • Phosphorilated OmpR concentration collapse}
  • Pops output


Green Emission

Description

Green Emission is composed of a series of enzymes that generate light by the oxidation of a substrate. Our sub-device has 6 enzymes (LuxA, LuxB, LuxC, LuxD, LuxE, LuxY), two catalyze the oxidation step (LuxA, LuxB), one adjusts the emission spectrum (LuxY), and three generate and recycle the substrate (LuxC, LuxD, LuxE). We plan on having the adjusting enzyme, as well as the 3 regenerating enzymes expressed constitutively. We would then only use the oxidation enzymes as reporters for whatever event we are observing.

While the oxidation per se does not generate light, it does generate an intermediate molecule in an electronically exited state. When said molecule returns to a basal energy state, a photon is released.

As you may imagine, these genes constitute an Operon. This is the Lux Operon from Vibrio fischeri.

See also the work of Edinburgh 2009.


Signaling Cascade

When our device recieves Pops, the following cascade will ensure:

  • Pops intput
  • Transcription of genes downstream of OmpF promoter: LuxA & LuxB
  • Oxidation of substrate
  • Photon output


Green Reception

Description

Green Reception is composed of a two-component system. Firstly, we have a sensing agent (CcaS). This protein shows two basal states, both with histidine kinase activities but each with an affinity for different substrates: a phenomenon known as photoconversion. We plan on using the Green phase regulator (CcaR) who happens to be a Transcription Factor.

When our regulator is in a phosphorilate state, it shows greater affinity for DNA. Thus it is active. The target promoter region has been recently identified. We thus plan on constructing our reporter genes under this promoter. Such a construction would be an <IF Light> logic gate. This system is quite similar to the EnvZ-OmpR system.

See also this paper.


Signaling Cascade

When our device is struck by green light, the following cascade will ensure:

  • Photon input
  • CcaS switches to Green conformation
  • Kinase activity starts
  • Phosphorilated CcaR concentration builds up
  • Pops output


Blue Emission

Description

Blue Emission is composed of a series of enzymes that generate light by the oxidation of a substrate. Our sub-device has 6 enzymes (LuxA, LuxB, LuxC, LuxD, LuxE, LumP), two catalyze the oxidation step (LuxA, LuxB), one adjusts the emission spectrum (LumP), and three generate and recycle the substrate (LuxC, LuxD, LuxE). We plan on having the adjusting enzyme, as well as the 3 regenerating enzymes expressed constitutively. We would then only use the oxidation enzymes as reporters for whatever event we are observing.

While the oxidation per se does not generate light, it does generate an intermediate molecule in an electronically exited state. When said molecule returns to a basal energy state, a photon is released. Likewise, LumP does not actually shift the spectrum, but the enzyme generates a substrate that does.

As you may imagine, these genes (sauf LumP) constitute an Operon. This is the Lux Operon from Vibrio fischeri.

See also the work of Edinburgh 2009.


Signaling Cascade

When our device receives Pops, the following cascade will ensure:

  • Pops input
  • Transcription of genes downstream of target promoter: LuxA & LuxB
  • Oxidation of substrate
  • Photon output


Blue Reception

Description

Blue Reception is composed of a slightly complicated system. Firstly, we have a quimeric sensing protein (LovTAP). This protein is composed of a sensing domain (a Light-Oxygen-Voltage domain) as well as the TAP protein from the Triptophan pathway. This protein will dimerize when struck by blue light. As a dimer, it shows greater affinity for DNA at the TrpO promoter. However, it acts as an inhibitor to transcription. Occupied TrpO is repressed while free TrpO is active.

Our construction is consequently based on an "inhibit the inhibitor" logic. By constructing our reporter genes under a repressed promoter (TetO), and having LovTAP repress the inhibitor of said promoter (TetR), we establish a direct <IF Light> logic gate.

See also LovTAP.


Signaling Cascade

When our device is struck by blue light, the following cascade will ensure:

  • Photon input
  • Lov-domain conformational change
  • LovTAP dimerization
  • TrpO promoter is repressed
  • Concentration of TetR collapses
  • TetO promoter is free
  • Pops output


Red Emission

Description

Red Emission is composed of mainly two enzymes. Our first enzyme (Luciferase) is a mutated form of the wild type enzyme found in Photinus pyralis. Our mutant is expected to glow red instead of the wild type blue-green. This enzyme catalyzes the oxidation reaction that yields light. The substrate for this reaction (Luciferin) is a most complicated molecule, and to our knowledge no one has ever managed to produce it within an E. coli chassis. Therefore, we need to inoculate the medium with luciferin to enable the reactions. However, recently a new enzyme was discovered (LRE) that recycles luciferin. We thus need only an initial inoculation with luciferin and from there on, the system is sufficiently autonomous.

We plan on using Luciferase as a reporter gene, while having LRE expressed constitutively.

For the Red Emission mutation, see this paper.

For the Red Emission protein, see this BioBrick part.


Signaling Cascade

  • Pops input
  • Luciferase downstream of TetO is transcribed
  • Oxidation of substrate
  • Photon output

iGEM

iGEM is the International Genetically Engineered Machines Competition, held each year at MIT and organized with support of the Parts Registry. See more here.

Synthetic Biology

This is defined as attempting to manipulate living objects as if they were man-made machines, specifically in terms of genetic engineering. See more here.

Genomics

We are students on the Genomic Sciences program at the Center for Genomic Sciences of the National Autonomous University of Mexico, campus Morelos. See more here.

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