Team:UNAM-Genomics Mexico/Project

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|Spanish=
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__NOTOC__
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<html><a href="http://2010.igem.org/Team:UNAM-Genomics_Mexico/es/Project"><img src="http://2010.igem.org/wiki/images/c/cc/UNAM-Genomics_Mexico_Flag_of_Mexico.svg.png" title="Español" height="20px"/></a></html>
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='''English'''=
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|English=
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__NOTOC__
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<html><a href="http://2010.igem.org/Team:UNAM-Genomics_Mexico/Project"><img src="http://2010.igem.org/wiki/images/2/28/UNAM-Genomics_Mexico_Flag_of_the_United_Kingdom.svg.png" title="English" height="20px" /></a></html>
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|French=
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<html><a href="http://2010.igem.org/Team:UNAM-Genomics_Mexico/fr/Project"><img src="http://2010.igem.org/wiki/images/b/b3/UNAM-Genomics_Mexico_Flag_of_France.svg.png" title="Français" height="20px" /></a></html>
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|Red_Content=
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__NOTOC__
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==Synthetic Biology==
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This is defined as attempting to manipulate living objects as if they were man-made machines, specifically in terms of genetic engineering. See more [[Team:UNAM-Genomics_Mexico/About/Synthetic_Biology|here]].
 +
 
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|Green_Content=
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__NOTOC__
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==iGEM==
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iGEM is the International Genetically Engineered Machines Competition, held each year at MIT and organized with support of the Parts Registry. See more [[Team:UNAM-Genomics_Mexico/About/iGEM|here]].
 +
 
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|Blue_Content=
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__NOTOC__
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==Genomics==
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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 [[Team:UNAM-Genomics_Mexico/About/CCG|here]].
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|Markup_Content=
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__NOTOC__
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__TOC__
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|Main_Content=
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__NOTOC__
== '''Overall project''' ==
== '''Overall project''' ==
 +
 +
 +
==='''The Idea'''===
Synthetic Biology has been enabling changes in all bio-domains, one such being
Synthetic Biology has been enabling changes in all bio-domains, one such being
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it is a natural step in the communications bridge between organic-based and
it is a natural step in the communications bridge between organic-based and
silicon-based systems, such as computers.
silicon-based systems, such as computers.
 +
 +
 +
==='''The Name'''===
 +
 +
As you might imagine, WiFi is a play on the popular IEEE 802.11 communications standard knows as Wi-Fi. Since our systems achieves the transfer of information without wires, it is thus wireless. As for fidelity, we shall see.
 +
 +
Before you begin wondering on Copyright issues, let us make two things clear:
 +
* Our name is WiFi, not the [http://www.wi-fi.org/ Wi-Fi Alliance]'s Wi-Fi (notice the hyphen).
 +
* Second, a quick search on the US Patents & Trademarks Office [http://tess2.uspto.gov/ Trademark Electronic Search System (TESS)] returned that WiFi was a trademark in 2006, but is now listed as "Dead". Therefore, we are not infringing copyright issues by using it in our system.
 +
== Project Details==
== Project Details==
 +
The process of transferring information from a sender entity to a receiver one through a determined channel is called communication. Biological entities have relied since time immemorial on chemical messengers to relay information; this holds true for multicellular organisms as well as for populations of unicellular organisms. Being chemical based, these messengers are constrained to a chemical system regardless of the scope of said system, eg: even far reaching messengers such as hormones are bound within the chemical system that is the human body.
The process of transferring information from a sender entity to a receiver one through a determined channel is called communication. Biological entities have relied since time immemorial on chemical messengers to relay information; this holds true for multicellular organisms as well as for populations of unicellular organisms. Being chemical based, these messengers are constrained to a chemical system regardless of the scope of said system, eg: even far reaching messengers such as hormones are bound within the chemical system that is the human body.
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Our ambicious implementation is based on well known systems, mainly bioluminescent proteins from Photinus pyralis and Vibrio fischeri, as well as photoactive receptors like Cyanobacteria cyanobacteriochromes and Light-Oxygen-Voltage domain quimeric proteins. We thus exploit the fact that cells already display primitive photo-communication, both within multicellular organisms as well as within populations of unicellular ones. Moreover, in our system the photonic information is transformed to and from chemical information within the chemical system that is an individual cell. Thus, the chemical barrier that is the membrane has ceased being a barrier to communication and is now a noise isolator. By decoupling the messenger from the chemical layer, we enable a brand new host of applications that were previously unavailable, ranging in domains from neurobiology, to cybernetic coupling, and even to biological telecommunications.
Our ambicious implementation is based on well known systems, mainly bioluminescent proteins from Photinus pyralis and Vibrio fischeri, as well as photoactive receptors like Cyanobacteria cyanobacteriochromes and Light-Oxygen-Voltage domain quimeric proteins. We thus exploit the fact that cells already display primitive photo-communication, both within multicellular organisms as well as within populations of unicellular ones. Moreover, in our system the photonic information is transformed to and from chemical information within the chemical system that is an individual cell. Thus, the chemical barrier that is the membrane has ceased being a barrier to communication and is now a noise isolator. By decoupling the messenger from the chemical layer, we enable a brand new host of applications that were previously unavailable, ranging in domains from neurobiology, to cybernetic coupling, and even to biological telecommunications.
-
=== Reception ===
 
-
We plan on using a cyanobacteria phytochrome-like domain coupled to an EnvZ kinase to sense incoming red light and translate it into a signal detectable by the cell, in this case phosphorilated OmpR. This brilliant system was a construction known as [http://partsregistry.org/Coliroid Coliroid] for the 2004 iGEM by the University of Texas at Austin and UCSF iGEM team. This system acts as an <IF ! LIGHT> logic gate. We are contemplating the use of distinct assemblies to obtain a direct <IF LIGHT> logic gate.
 
-
Our second sensing device is based on another BioBrick Part: [http://partsregistry.org/Part:BBa_K191003 LOVtap]. This TF dimerizes when struck by blue light, thus activating the transcription of a determined promoter's coding sequence.
+
==Module Logic==
 +
 
 +
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.
 +
 
 +
 
 +
==Individual Modules==
 +
 
 +
To learn more about the modules, here's a short description on them.
 +
 
 +
<html>
 +
<form name="dropmsgform">
 +
<select name="dropmsgoption" size="1" style="width:300" onChange="expandone()">
 +
    <option selected>Red Reception</option>
 +
    <option>Green Emission</option>
 +
    <option>Green Reception</option>
 +
    <option>Blue Emission</option>
 +
    <option>Blue Reception</option>
 +
    <option>Red Emission</option>
 +
</select>
 +
<br>
 +
 
 +
 
 +
<div id="dropmsg0" class="dropcontent">
 +
</html>
 +
===Red Reception===
 +
 
 +
[[Image:Cph8.jpg|550px|center]]
 +
 
 +
 
 +
====Description====
 +
 
 +
The cyanobacterial phytochrome (Cph1) fused to the EnvZ histidine kinase domain from E.coli, makes up the chimaeric photoreceptor Cph8 constructed by Levskaya and collaborators. This photosensor requires a specific linear tetrapyrrole cofactor, phycocyanobilin (PCB), to detect red light.  This 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.
 +
 
 +
The light responsive domain (Cph1) has maximal response to light near 660nm. 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, [http://partsregistry.org/Coliroid 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
 +
 
 +
====References====
 +
 
 +
Levskaya, A., Chevalier, A. A., & Tabor, J. J. (2005). Engineering Escherichia coli to see light. Nature, 438(7067), 442. doi: 10.1038/438442a.
 +
 
 +
<html>
 +
</div>
 +
<div id="dropmsg1" class="dropcontent">
 +
</html>
 +
 
 +
 
 +
===Green Emission===
 +
 
 +
[[Image:LuxY.jpg|350px|center]]
 +
 
 +
====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 (YFP), and three generate and recycle the substrate (LuxC, LuxD, LuxE). This complex converts fatty acids to aldehydes which are in turn used as a substrate by bacterial luciferase to emit light. 
 +
 
 +
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.
 +
 
 +
This light emission system has been taken from the bacterium Vibrio fischeri.
 +
The natural emission spectrum of Vibrio fischeri is blue, nevertheless this strain must emit green light, the spectrum shift is achieved by means of an "antenna" protein called YFP (taken from Vibrio fischeri strain Y-1) which receives the light emitted by bacterial luciferase and then emits light of a different wavelength (yellow in this case), YFP uses NADPH as a substrate.
 +
 
 +
 
 +
See also the work of [http://2009.igem.org/Team:Edinburgh/biology(biobricks) Edinburgh 2009].
 +
 
 +
 
 +
====Signaling Cascade====
 +
 
 +
When our device recieves Pops, the following cascade will ensure:
 +
* Pops intput
 +
* Transcription of genes downstream of promoter: LuxA & LuxB
 +
* Oxidation of substrate
 +
* Photon output
 +
 
 +
<html>
 +
</div>
 +
<div id="dropmsg2" class="dropcontent">
 +
</html>
 +
 
 +
===Green Reception===
 +
 
 +
[[Image:CcaS.jpg|550px|center]]
 +
 
 +
====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 two-component system has been described in Synechocystis sp. PCC 6803, and due to the homology relationship with
 +
the EnvZ-OmpR system, it might possibly be functional in E.coli.
 +
 
 +
See also [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474522/ 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
 +
 
 +
<html>
 +
</div>
 +
<div id="dropmsg3" class="dropcontent">
 +
</html>
 +
 
 +
===Blue Emission===
 +
 
 +
[[Image:Lumazine.jpg|350px|center]]
 +
 
 +
 
 +
====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 [http://2009.igem.org/Team:Edinburgh/biology(biobricks) 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
 +
 
 +
<html>
 +
</div>
 +
<div id="dropmsg4" class="dropcontent">
 +
</html>
 +
 
 +
===Blue Reception===
 +
 
 +
===LovTAP===
 +
 
 +
[[Image:LovTAP_Genomics.jpg|550px|center]]
 +
 
 +
====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 DNA-binding domain of the TrpR transcriptional repressor from the Triptophan pathway.
 +
 
 +
LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm.
 +
 
 +
 
 +
Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes  DNA-binding affinity at the trpL promoter and LovTAP can then bind to DNA as an homodimer, repressing the transcription of the genes downstream of the promoter.
 +
 
 +
In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.
 +
 
 +
Our construction is consequently based on an "inhibit the inhibitor" logic. By constructing our reporter genes under a repressed promoter (cI binding site), and having LovTAP repress the inhibitor of said promoter (cI lambda repressor), 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
 +
* trpL promoter is repressed
 +
* Concentration of cI repressor collapses
 +
* trpL promoter is free
 +
* Pops output
 +
 
 +
====References====
 +
 
 +
Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.
 +
 
 +
STRICKLAND, D. (2009). NEW APPROACHES TO THE DESIGN OF ALLOSTERIC PROTEINS.
 +
 
 +
 
 +
===YcgF/YcgE blue reception system===
 +
 
 +
[[Image:Bluepromoter.jpg| 550px| center]]
 +
 
 +
====Description====
-
Our final input device is based on a newfound OmpR-like system of Synechocystis. This system uses a sensing protein (CcaS) than shows kinase activity when struck by green light. It then proceeds to phosphorilate a regulatroy TF (CcaR). This TF then starts transcription of its associated promoter's coding sequence.
+
The YcgF/YcgE system is based on the action of the repressor YcgE, which is bound to
 +
the promoter region when there is no blue light, thus inhibiting the transcription of any
 +
gene downstream this promoter.
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=== Emission ===
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In the presence of blue light, YcgF dimerizes and now it has a great affinity for YcgE,
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We plan on using a mutated version of Photinus pyralis luciferase to generate red light. In addition, we will be using a newfound enzyme called Luciferin Regenerating Enzyme to recycle said substrate.
+
clearing the promoter and allowing transcription to proceed.
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For the blue and green emissions, we will be using the Lux Operon from Vibrio fischeri. We plan on taking advantage of the lumazine BioBrick part to generate a blue-shifted light (around 450nm), as well as the LuxY of strain Y-1 to generate a red-shifted light (around 550nm).
+
It is reported that the response of this promoter is weak in comparison to some others
 +
standard strong promoters registered in the Registry of Standard Biological Parts.
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=== Application ===
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====Signaling Cascade====
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While glowing bacteria are nothing new, photo-communicating bacteria are something quite rare. We plan on using this advantage on several applications as a proof-of-principle. This may include an oscillator, a bio-cable, cell-phone-using bacteria, among others...
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-
== Results ==
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* Photon input
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(None yet... but check back soon!)
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* YcgF dimerization
 +
* Binding of YcgF dimer with YcgE.
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* Blue promoter is free
 +
* Pops output
 +
<html>
 +
</div>
 +
<div id="dropmsg5" class="dropcontent">
 +
</html>
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='''Español'''=
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===Red Emission===
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== '''Descripcion general del proyecto''' ==
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[[Image:REDLuciferase.jpg|350px|center]]
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La Biología Sintética ha permitido cambios en todo ámbito biologico, uno de estos es la
+
====Description====
-
comunicación. La comunicación celular se ha basado desde tiempos inmemoriales en
+
-
mensajeros químicos usados para intercambiar información. Como tal, estos mensajeros, independientemente
+
-
de su alcance, se ven limitados a un sistema químico, incluyendo hasta esos
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-
mensajeros de largo alcanze, como las hormonas se vinculan a un sistema químico (cuerpo humano).
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-
Sin embargo, este modo de comunicación está a punto de cambiar.
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-
En este proyecto, nuestro objetivo es hacer que la barrera química  de la comunicación celular
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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.
-
se vuelva obsoleta al usar mensajeros no químicos: fotones. Estos transportaran
+
-
información entre células que han sido diseñadas para detectar y emitir luz,
+
-
creando así un sistema de comunicación basado en fotones inter-celulares.
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-
Estos mensajeros son producidos a través de reacciones bioluminiscentes y
+
We plan on using Luciferase as a reporter gene, while having LRE expressed constitutively.
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son capaces de atravesar múltiples entornos. Por ende, esta capacidad permite
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-
la propagación de información más allá de las restricciones químicas, biológicas y e incluso
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-
espaciales. Como el mensajero es efectivamente libre de la barrera química,
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-
es un paso natural en el puente de comunicación entre los sistemas de base orgánica y
+
-
aquellos basados en el silicon, como las computadoras.
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-
== Detalles del proyecto==
+
For the Red Emission mutation, see [http://dx.doi.org/10.1016/j.ab.2005.07.015 this paper].
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El proceso de transferir información de un emisor a un receptor a través de un canal determinado se llama comunicación. Entidades biológicas se han basado desde tiempos inmemoriales en mensajeros químicos para transmitir información; Válido para los organismos multicelulares, así como para las poblaciones de organismos unicelulares. Al ser compuestos químicos, estos mensajeros se ven limitados al alcanze de dicho sistema, por ejemplo: Aun mensajeros de largo alzanze, como las hormonas, estan ligadas al sistema quimico (cuerpo humano).
+
-
En este proyecto, nuestra meta es hacer que la barrera química  de la comunicación celular
+
For the Red Emission protein, see [http://partsregistry.org/Part:BBa_I712019:Design this BioBrick part].
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se vuelva obsoleta.Esto se traduce al uso de mensajeros no químicos, en este caso, los fotones.
+
-
Nuestro canal entonces es la luz hecha apartir de paquetes de fotones o cuantas de energía, que transportaran información de los remitentes a los receptores, cruzando asi murallas quimicas.
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-
En consecuencia, nuestro sistema de comunicación ya no es contenida dentro de un sistema químico, pero dentro de uno físico, es decir: debe de existir un canal fisico por el cual los fotones puedan ser transportados.
+
-
Este canal físico puede variar desde algo tan sofisticado como un sistema de enlaces basado en microcontroladores electrónicos, hasta algo tan simple como el aire (o el vacio!!).
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-
Sin embargo, esta capa física debe ser impermeable a la señalización de los componentes químicos. Por tanto, el sistema de señalización química no se vería afectada por el canal físico, y viceversa.
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-
En consecuencia, el intercambio de información a través de medios físicos es suficientemente independiente de la información codificada en las vías del sistema químico endógeno. En otras palabras, es extraordinariamente no invasivo.
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-
Como bono adicional, nuestras entidades receptoras son fácilmente transformadas en entidades emisoras.
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-
Así, usando nuestras células como chasis de procesamiento de información, podemos ampliar la capade comunicaciones.
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-
Nosotros podemos hacer nuestro sistema efectivo donde la informacion es:
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-
* Codificada y enviada por un emisor
+
-
* Recibida y decodificada por un receptor
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-
* Procesada, transformada y transmitida sucesivamente
+
-
Our ambicious implementation is based on well known systems, mainly bioluminescent proteins from Photinus pyralis and Vibrio fischeri, as well as photoactive receptors like ---------------------- and Light-Oxygen-Voltage domain quimeric proteins. We thus exploit the fact that cells already display primitive photo-communication, both within multicellular organisms as well as within populations of unicellular ones. Moreover, in our system the photonic information is transformed to and from chemical information within the chemical system that is an individual cell. Thus, the chemical barrier that is the membrane has ceased being a barrier to communication and is now a noise isolator. By decoupling the messenger from the chemical layer, we enable a brand new host of applications that were previously unavailable, ranging in domains from neurobiology, to cybernetic coupling, and even to biological telecommunications.
 
-
=== Recepción ===
+
====Signaling Cascade====
-
Queremos usar un complejo quimérico unido a la cinasa EnvZ para captar la luz roja y transladarla a una señal detectable para la célula, en este caso OMPR fosforilado. Este espectacular sistema es ampliamente conocido como [http://partsregistry.org/Coliroid Coliroid] en el iGEM 2004 creado por el equipo de la universidad de Texas y el quipo de UCSF. El siguiente sistema funciona como una puerta logica ''NO luz''. Nosotros contemplamos un uso distinto de este receptor para obtener directamente una puerta logica del tipo ''Si hay luz''.
+
-
Nuestro segundo dispositivo de detección esta basado en la bioparte: [http://partsregistry.org/Part:BBa_K191003 LOVtap]. Este factor de transcripción se dimeriza al ser excitado por la luz azul, conllevando así a la transcripción de una determinada secuencia del promotor. Por otro lado, estamos viendo la posibilidad del uso del promotor azul <new>.
+
* Pops input
 +
* Luciferase downstream of promoter is transcribed
 +
* Oxidation of substrate
 +
* Photon output
-
Por ultimo, y no así menos importante, nuestro dispositivo de entrada final se basa en un nuevo descubrimiento en un sistema parecido al de OmpR en Synechocystis. Este sistema usa una proteina sensadora(CcaS) que pone al descubierto un motivo de cinasa al ser irradiado con luz verde. Lo que procede a fosforilar el factor de transcripcion regulatorio ''CcaR''. Y así este TF activa la transcripcion.
+
<html>
 +
</div>
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=== Emisión ===
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Nosotros planteamos el uso de una versión mutada de la luciferasa de Photinus pyralis para generar un rojo difuso. En adición, pretendemos usar la recién descubierta enzima regeneradora de luciferina LRE, llamada así por sus siglas en ingles.
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Para las emisiones de verde y azul pretendemos el uso del operón LUX de Vibrio Fisheri. Tomaremos de ventaja el biobrick de la proteína lumazina para generar una luz azul desfasada en el espectro, y de misma manera, el uso de la cepa LuxY para generar un espectro desfasado equivalente al verde.
 
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=== Aplicaciones ===
 
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Mientras que las bacterias luminosas no son nuevas, las bacterias foto-comunicantes son raras. Nosotros planeamos usar esta ventaja en diferentes aplicaciones basadas en el mismo principio. Incluyendo osciladores, bio-cables, telefonos celulares usando bacterias, entre otras...
 
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== Resultados ==
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(Aun no... pero, mantente en contacto!)
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Latest revision as of 09:20, 27 October 2010



Overall project

The Idea

Synthetic Biology has been enabling changes in all bio-domains, one such being communication. Cellular communication has relied since time immemorial on chemical messengers to exchange information. As such, these messengers regardless of their scope, are constrained to a chemical system; even far reaching messengers such as hormones are bound within the chemical system that is the human body. But this mode is about to change.

In this project, our goal is to render the chemical barrier deprecated by using a non-chemical messenger: photons. These will transport information between cells that have been designed to sense and emit light, thus creating a photon-based inter-cellular communication system.

These messengers are produced through bio-luminescent reactions, and are quite capable of traversing multiple environments. Consecuently, this enables the propagation of information beyond the chemical, biological and even spatial restrictions. As the messenger is effectively decoupled from the chemical layer, it is a natural step in the communications bridge between organic-based and silicon-based systems, such as computers.


The Name

As you might imagine, WiFi is a play on the popular IEEE 802.11 communications standard knows as Wi-Fi. Since our systems achieves the transfer of information without wires, it is thus wireless. As for fidelity, we shall see.

Before you begin wondering on Copyright issues, let us make two things clear:

  • Our name is WiFi, not the Wi-Fi Alliance's Wi-Fi (notice the hyphen).
  • Second, a quick search on the US Patents & Trademarks Office Trademark Electronic Search System (TESS) returned that WiFi was a trademark in 2006, but is now listed as "Dead". Therefore, we are not infringing copyright issues by using it in our system.


Project Details

The process of transferring information from a sender entity to a receiver one through a determined channel is called communication. Biological entities have relied since time immemorial on chemical messengers to relay information; this holds true for multicellular organisms as well as for populations of unicellular organisms. Being chemical based, these messengers are constrained to a chemical system regardless of the scope of said system, eg: even far reaching messengers such as hormones are bound within the chemical system that is the human body.

In this project, our goal is to render the chemical barrier deprecated by enabling chemical-free communication. This has been translated to the implementation of a non-chemical messenger, in this case, photons. Our channel is thus light based; packages of photons, or energy quanta, will transport information from senders to receivers, effectively bypassing most chemical barriers in-between. Consequently, our communicating system is no longer contained within a chemical system, but within a physical one, ie: there must remain a physical channel where photons can be transported. This physical channel may range from something as sophisticated as a microcontroler-based electronic relay system, to something as simple as vacuum (or void). However, this physical layer proves very well to be impervious to most chemical signaling. Ergo, the chemical system's signaling would remain unaffected by the physical channel, and vice versa. In consequence, the exchange of information through physical means is sufficiently independent from the information encoded in the system's endogenous chemical pathways. In other words, it is extraordinarily uninvasive. As an added bonus, our receiver entities are easily transformed into emitter entities. Thus, by using our cells as information processing chassis, we can expand the communications layer. We can effectively render our system one where information is:

  • Encoded and sent by an emitter
  • Recieved and decoded by a receiver
  • Plus processed, transformed, and relayed forth

Our ambicious implementation is based on well known systems, mainly bioluminescent proteins from Photinus pyralis and Vibrio fischeri, as well as photoactive receptors like Cyanobacteria cyanobacteriochromes and Light-Oxygen-Voltage domain quimeric proteins. We thus exploit the fact that cells already display primitive photo-communication, both within multicellular organisms as well as within populations of unicellular ones. Moreover, in our system the photonic information is transformed to and from chemical information within the chemical system that is an individual cell. Thus, the chemical barrier that is the membrane has ceased being a barrier to communication and is now a noise isolator. By decoupling the messenger from the chemical layer, we enable a brand new host of applications that were previously unavailable, ranging in domains from neurobiology, to cybernetic coupling, and even to biological telecommunications.


Module Logic

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.


Individual Modules

To learn more about the modules, here's a short description on them.


Red Reception

Cph8.jpg


Description

The cyanobacterial phytochrome (Cph1) fused to the EnvZ histidine kinase domain from E.coli, makes up the chimaeric photoreceptor Cph8 constructed by Levskaya and collaborators. This photosensor requires a specific linear tetrapyrrole cofactor, phycocyanobilin (PCB), to detect red light. This 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.

The light responsive domain (Cph1) has maximal response to light near 660nm. 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

References

Levskaya, A., Chevalier, A. A., & Tabor, J. J. (2005). Engineering Escherichia coli to see light. Nature, 438(7067), 442. doi: 10.1038/438442a.


Green Emission

LuxY.jpg

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 (YFP), and three generate and recycle the substrate (LuxC, LuxD, LuxE). This complex converts fatty acids to aldehydes which are in turn used as a substrate by bacterial luciferase to emit light.

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.

This light emission system has been taken from the bacterium Vibrio fischeri. The natural emission spectrum of Vibrio fischeri is blue, nevertheless this strain must emit green light, the spectrum shift is achieved by means of an "antenna" protein called YFP (taken from Vibrio fischeri strain Y-1) which receives the light emitted by bacterial luciferase and then emits light of a different wavelength (yellow in this case), YFP uses NADPH as a substrate.


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 promoter: LuxA & LuxB
  • Oxidation of substrate
  • Photon output

Green Reception

CcaS.jpg

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 two-component system has been described in Synechocystis sp. PCC 6803, and due to the homology relationship with the EnvZ-OmpR system, it might possibly be functional in E.coli.

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

Lumazine.jpg


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

LovTAP

LovTAP Genomics.jpg

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 DNA-binding domain of the TrpR transcriptional repressor from the Triptophan pathway.

LOV domains bind a flavin-mononucleotide (FMN) or flavin-adenine-dinucleotide (FAD) cofactor, which are used in a wide variety of metabolic pathways as cofactors in redox reactions and are available in most organisms. The cofactor has a broad absorption spectrum, with a maximum at 450 nm.


Under the presence of light, absorption of a photon leads to the formation of a covalent adduct between the flavin mononucleotide (FMN) cofactor and a conserved cysteine residue in the AsLOV2 domain, which results in conformational rearrangements in LovTAP. This change impacts the affinity of the shared helix for the two domains: disrupting the contacts between the shared helix and the LOV domain and enabling the association of the shared helix with the TrpR domain, which establishes DNA-binding affinity at the trpL promoter and LovTAP can then bind to DNA as an homodimer, repressing the transcription of the genes downstream of the promoter.

In the dark, when the shared helix contacts the LOV domain, the TrpR domain's DNA-binding affinity decreases and LovTAP is in an inactive conformation.

Our construction is consequently based on an "inhibit the inhibitor" logic. By constructing our reporter genes under a repressed promoter (cI binding site), and having LovTAP repress the inhibitor of said promoter (cI lambda repressor), 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
  • trpL promoter is repressed
  • Concentration of cI repressor collapses
  • trpL promoter is free
  • Pops output

References

Strickland, D., Moffat, K., & Sosnick, T. (2008). Light-activated DNA binding in a designed allosteric protein. Proceedings of the National Academy of Sciences, 105(31), 10709. National Acad Sciences. Retrieved from http://www.pnas.org/content/105/31/10709.full.

STRICKLAND, D. (2009). NEW APPROACHES TO THE DESIGN OF ALLOSTERIC PROTEINS.


YcgF/YcgE blue reception system

Bluepromoter.jpg

Description

The YcgF/YcgE system is based on the action of the repressor YcgE, which is bound to the promoter region when there is no blue light, thus inhibiting the transcription of any gene downstream this promoter.

In the presence of blue light, YcgF dimerizes and now it has a great affinity for YcgE, clearing the promoter and allowing transcription to proceed.

It is reported that the response of this promoter is weak in comparison to some others standard strong promoters registered in the Registry of Standard Biological Parts.

Signaling Cascade

  • Photon input
  • YcgF dimerization
  • Binding of YcgF dimer with YcgE.
  • Blue promoter is free
  • Pops output

Red Emission

REDLuciferase.jpg

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 promoter 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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