Team:UNAM-Genomics Mexico/Project

From 2010.igem.org

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===Red Reception===
===Red Reception===
 +
 +
[[Image:Cph8.jpg|550px|center]]
====Description====
====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.
+
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.
-
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.
+
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.
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.
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* Phosphorilated OmpR concentration collapse
* Phosphorilated OmpR concentration collapse
* Pops output
* 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.
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===Green Emission===
===Green Emission===
 +
[[Image:LuxY.jpg|350px|center]]
====Description====
====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.
+
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.
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.
+
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].
See also the work of [http://2009.igem.org/Team:Edinburgh/biology(biobricks) Edinburgh 2009].
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===Green Reception===
===Green Reception===
 +
[[Image:CcaS.jpg|550px|center]]
====Description====
====Description====
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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.
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.
+
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].
See also [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2474522/ this paper].
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===Blue Emission===
===Blue Emission===
 +
 +
[[Image:Lumazine.jpg|350px|center]]
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===Blue Reception===
===Blue Reception===
 +
===LovTAP===
 +
 +
[[Image:LovTAP_Genomics.jpg|550px|center]]
====Description====
====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.
+
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.  
-
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.
+
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.  
-
See also [http://partsregistry.org/Part:BBa_K191003 LovTAP].
 
 +
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====
====Signaling Cascade====
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* Lov-domain conformational change
* Lov-domain conformational change
* LovTAP dimerization
* LovTAP dimerization
-
* TrpO promoter is repressed
+
* trpL promoter is repressed
-
* Concentration of TetR collapses
+
* Concentration of cI repressor collapses
-
* TetO promoter is free
+
* 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====
 +
 
 +
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
* Pops output
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===Red Emission===
===Red Emission===
 +
[[Image:REDLuciferase.jpg|350px|center]]
====Description====
====Description====

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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