Paris/Perspectives

From 2007.igem.org

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(System improvement through directed evolution)
 
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SMB is composed of a germ line and a soma. Germ cells are responsible of the reproduction while the sterile somatic cells, are unable generate a population independently themselves but are essential for the germ line, by exporting Dap compound. The special feature of our system, the coexistence of two independent cell types, one dedicated to reproduction and the other sterile, render it a potentially interesting tool for synthetic biology.  
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SMB is composed of a germline and a soma. Germ cells are responsible for reproduction while the sterile somatic cells, are essential for the germline as the soma exports the DAP compound, supporting germline growth. The special feature of our system, the coexistence of two independent cell types, one dedicated to reproduction and the other sterile, makes it a potentially interesting tool for synthetic biology.  
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We envision two immediate applications to the Synthetic Multicellular Bacterium. It could be used as a “metabolic plant” or as “security device”
 
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The SMB could also be used to explore basic questions in fundamental biology. If submitted to long time growth, in a chemostat for instance,  the SMB could become a model to study how the genome of a cell can evolve leading to changes that do not directly affect the cells phenotype. This system could mimic evolutionary phenomena that are typicall of multicellular organisms.
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== System improvement through directed evolution ==
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= System improvement through directed evolution =
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The engineered SMB may be further tuned by laboratory evolution. A key advantage of the system is that selection pressure could be tuned to either force faster reproduction (e.g., improving DAP production and/or export from the soma or alternatively for a more efficient use of DAP within the germline) or for improvement of soma-specific functions (see below). In other words, the soma-germline SMB dichotomy provides us with the possibility to select for functions that are otherwise deleterious for the unicellular organism.
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==Applications==
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=Applications=
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We envision two immediate applications to the Synthetic Multicellular Bacterium. It could be used as a “metabolic plant” or as “security device”
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=== Decoupling reproduction faculty and transgene expression ===
 
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SMB is made up with a germ line and somatic line. Germ cells are responsible for the reproduction of the organism and the others, the sterile somatic cells, are unable to build a new organism and themselves but are essential for the germ line, exporting Dap compound. That is the basic property of our SMB and this is the key to original approaches.
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== Decoupling reproduction faculty and transgene expression ==
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Indeed the reproductive faculty in our system is independent with DAP production. Either the cell produces Dap (somatic cell) or it is able to divide. We can also imagine adding a transgene downstream the dapA gene. What would be the consequence? The somatic cell would be able to synthesize a new gene product. Knowing the soma is not able to divide and reproduce the organism, we are able to disrupt the connection between the reproduction of the system and the expression of the transgene leading to interesting applications.  
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In the SMB system, any given gene or gene circuit can be selectively expressed in germ cells or somatic cells. Knowing the soma is unreplicative, we are disrupting the connection between the reproduction of the system and the expression of the transgene, leading to potentially interesting applications.  
<br><br>
<br><br>
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We want to develop two sides of this system: metabolic engineering and secured genetic modified organism (GMO).
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We would like to develop two extensions to the SMB : metabolic engineering and secured genetically modified organisms for environmental release.
===Security device===
===Security device===
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'''secure isolation of the soma => release in the environment'''
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We came up with the idea of modifying the synthetic organism in order to allow, on demand, the full differentiation of the germline into soma in a secured fashion. Why would we want to do that?
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This possibility is to be considered in the face of biohazard risks. Release of GM organisms in the environment poses ethical as well as technical questions. “Biological security” should be a prime concern in synthetic biology.
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We also came up with the idea of modifying the synthetic organism in order to allow on demand, full differentiation of the germ line into soma in a secured fashion. Why would we want to do that? This possibility is to be considered in the face of biohazard risks. Release of GM organisms in the environment poses ethical as well as technical questions. “Biological security” should be a prime concern in synthetic biology.
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If a task is to be transiently performed by a GM organism in the open environment, a major problem is persistence of the GM organism, with potential risks of proliferation and transgene dissemination. A possible solution, already used for some GM crops, is to render the organisms sterile. Can this solution be implemented for GM bacteria, in bioremediation systems for instance? Using a modified version of our SMB, the answer is yes. The SMB comprises two cell types, one being the soma, a group of cells that have a longer lifetime than wild type E.coli cells & are unable to replicate. The soma cells have the required characteristics for transient use in open environment.  
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If a task is to be transiently performed by a GM organism in the open environment, a major problem is persistence of the GM organism, with potential risks of proliferation and transgene dissemination. A possible solution, already used for some GM crops, is to render the organisms sterile. Can this solution be implemented for GM bacteria, in bioremediation systems for instance? Using a modified version of our SMB, the answer is yes. The SMB comprises two cell types, one being the soma, a group of cells that have a longer lifetime than wt E.coli cells & are unable to replicate. The soma cells have the required characteristics for transient use in open environment.
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The next consideration is then: can full differentiation be induced in the SMB? In order to achieve this, two events should be initiated:
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A next consideration is then: can full differentiation be induced in the SMB? In order to achieve this, two events should be initiated:
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* Induction of a massive differentiation of the germ line into soma cell
* Induction of a massive differentiation of the germ line into soma cell
* Followed by selective death of the persistent germ line cells.
* Followed by selective death of the persistent germ line cells.
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For details on proposed technical solutions to this challenge, [[see here]]
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For details on proposed technical solutions to this challenge, [[Security device|'''SEE HERE''']]
===Metabolic engineering===
===Metabolic engineering===
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A bacterial multicellular organism could allow optimizing the production of compounds deleterious to the cell. If you try to optimize the production of such a compound in a classical bacteria (E.coli for instance), you will reach a trade off between the production of your compound and the growth of the cell because the reproduction capacity is linked to the expression of the toxic transgene. The synthetic organism could in part bypass this problem by partially decoupling growth from synthesis of the exogenous compound. It could indeed be modified so that the soma only will produce the noxious molecule and the germ line will multiply. If there is a way to screen for the production of this molecule, we can then select the germ line whose soma would have the best yield. As long as the production doesn't impair too much the capacity of the soma to feed the germ line, the optimization can go on. Thus, there is also a trade off in this case, but it might very well be more favorable to the optimization than the trade off between production and growth. At least, this is worth testing.  
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A bacterial multicellular organism could allow optimizing the production of compounds deleterious to the cell. If one attempts to optimize the production of such a compound in a classical bacteria (E.coli for instance), trade-off appears between the production of your compound and the growth of the cell. The synthetic multicellular organism could in part bypass this problem by partially decoupling growth from synthesis of the exogenous compound. It could indeed be modified so that only the soma produces the noxious molecule. If a mean exists to screen for maximum production of this molecule, you could then select the germline whose soma would have the best exocompound production yield. As long as the production doesn't impair too much the capacity of the soma to feed the germline, the optimization can go on. Thus, a trade-off also exists in this case, but it would very well less stringent that in the wild type E.coli case.  
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Of course, all this is only possible if the germ line is not affected by the production of the soma. Compounds that could be optimized in this way are thus constrained to molecules that can be noxious in the cell, but that do not affect it to much if in the medium.
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Of course, all this is only possible if the germ line is not affected by the products released by the soma. Compounds that could be optimized in this way are thus constrained to molecules that can be noxious if accumulated in the cell, but that do not affect it too much if in the medium. For instance, if the product can be gradually filtered out of the medium.
== E. Colight: towards a new slim diet ==  
== E. Colight: towards a new slim diet ==  
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=== Project ===
=== Project ===
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Finally, the last part of our project would be to bring together the synthetic organism, the optimization of a compound noxious to the bacteria, and the security device. To do so, we'd like to optimize the production of triglycerides in the soma cells of our synthetic organism. We would then be able to differentiate all the germ line of the synthetic organism into soma in a secured way. Those super triglycerides-producing-not-able-to-divide cells could then be ingested. The fatty acids they would stock would be as many fatty acids you will not absorb! Eat fat, don't get fat! <br>
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In an attempt to integrate all the proposed applications of the SMB, we advanced towards the construction of Ecolight.  Bringing together the synthetic organism (SMB), noxious compound synthesis ("metabolic plant"), and the security device. This would include optimizing the production of triglycerides in the soma cells of our synthetic multicellular bacterium. Soma isolation according to the "security device" would be included. The super triglycerides-producing-not-able-to-divide soma cells could then be ingested. The fatty acids they would store would be as many fatty acids you will not absorb! Eat fat, don't get fat! <br>
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<br>
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Triglycerides are made of a molecule of glycerol esterified by three fatty acids. When ingested they are hydrolysed by lipase in the stomach and the duodenum, into glycerol and free fatty acids. Enterocytes are only able to absorb free fatty acids and glycerol and recombined them in the cytoplasm into triglycerides that will be free in the lymphatic system then in the blood within fatty vesicles called chylomicrons.
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<br><br>
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When we are becoming fat, it is because we have more input than output energy, lipid energy. Knowing that gut is full with bacteria forming the gut microflora (we have 10<sup>13</sup> of our cells in our body and 10<sup>14</sup> bacteria with the majority in the gut!), we could imaging having bacterium able to absorb fatty acids and form triglycerides intracellular inclusion. These triglycerides are not able to be absorbed by enterocytes! Input lipid is decreased! Eat fat, don't get fat!
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<br><br>
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In fact a drug, orlistat, already exists and shows that the concept of decreasing the lipid input works. It comes from a bacterial lipase inhibitor (from Streptomyces toxytricini). It is able to inhibit pancreatic lipase and is used to cure obese people and type 2 diabetes with hypocaloric cure. At the standard prescription dose of 120 mg three times daily before meals, orlistat prevents approximately 30% of dietary fat from being absorbed (Thomson PDR, 2006).
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<br><br>
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E.coli is the most used bacterium in synthetic biology and... belongs to the gut microflora! We could also imagine genetically engineering E. coli called E. colight to stock triglycerides into inclusions! Knowing that 40% of E.coli is renewed every day, these triglyceride-fulled bacteria will leave the gut with faeces! With the security device described above and derived from our SMB we should have a safe GMO with two hours life expectancy. This short action could be counterpart by repetitive ingestion of E. colight (yoghurts for example).
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[[Image: ecolight1.jpg|center|TG synthesis|400px]]
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E.colight project is also made up with 2 independent parts. First we have to create the SMB to implement the secured module. Second E.coli has to absorb free fatty acid and to synthesize triglycerides in the cytoplasm.
E.colight project is also made up with 2 independent parts. First we have to create the SMB to implement the secured module. Second E.coli has to absorb free fatty acid and to synthesize triglycerides in the cytoplasm.
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== Triglyceride synthesis ==
 
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See Results: [[Paris/Results#DGAT_cloning_and_triglyceride_synthesis_in_E._Coli|TG synthesis]]
 
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== Secured device ==
 
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See [[Paris/Perspectives#Security_device|Security device]] for the concept. The final contruction would be this one: <br>
 
[[Image: securitydevice4.jpg|center|600px]]
[[Image: securitydevice4.jpg|center|600px]]
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== Another biomedical applications ==
 
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Crohn disease and hemorrhagic recto-colitis are both autoimmune diseases located in the intestine. These diseases are thought to be caused by tolerance disruption of the body to its intestinal flora leading to abnormal inflammatory reponses. For example proinflammatory cytokines are produced and generate intestine lesions responsible of clinical symptoms.  New therapies are now targeting inflammatory cytokines. We could also imagine producing IL10 or IL11 in our secured SMB. These anti-inflammatory cytokines could also limit the extension of inflammatory lesions.<br><br>
 
'''References:''' <br>
'''References:''' <br>

Latest revision as of 00:52, 27 October 2007


SMB is composed of a germline and a soma. Germ cells are responsible for reproduction while the sterile somatic cells, are essential for the germline as the soma exports the DAP compound, supporting germline growth. The special feature of our system, the coexistence of two independent cell types, one dedicated to reproduction and the other sterile, makes it a potentially interesting tool for synthetic biology.


The SMB could also be used to explore basic questions in fundamental biology. If submitted to long time growth, in a chemostat for instance, the SMB could become a model to study how the genome of a cell can evolve leading to changes that do not directly affect the cells phenotype. This system could mimic evolutionary phenomena that are typicall of multicellular organisms.


Contents

System improvement through directed evolution

The engineered SMB may be further tuned by laboratory evolution. A key advantage of the system is that selection pressure could be tuned to either force faster reproduction (e.g., improving DAP production and/or export from the soma or alternatively for a more efficient use of DAP within the germline) or for improvement of soma-specific functions (see below). In other words, the soma-germline SMB dichotomy provides us with the possibility to select for functions that are otherwise deleterious for the unicellular organism.

Applications

We envision two immediate applications to the Synthetic Multicellular Bacterium. It could be used as a “metabolic plant” or as “security device”


Decoupling reproduction faculty and transgene expression

In the SMB system, any given gene or gene circuit can be selectively expressed in germ cells or somatic cells. Knowing the soma is unreplicative, we are disrupting the connection between the reproduction of the system and the expression of the transgene, leading to potentially interesting applications.

We would like to develop two extensions to the SMB : metabolic engineering and secured genetically modified organisms for environmental release.

Security device

We came up with the idea of modifying the synthetic organism in order to allow, on demand, the full differentiation of the germline into soma in a secured fashion. Why would we want to do that? This possibility is to be considered in the face of biohazard risks. Release of GM organisms in the environment poses ethical as well as technical questions. “Biological security” should be a prime concern in synthetic biology.

If a task is to be transiently performed by a GM organism in the open environment, a major problem is persistence of the GM organism, with potential risks of proliferation and transgene dissemination. A possible solution, already used for some GM crops, is to render the organisms sterile. Can this solution be implemented for GM bacteria, in bioremediation systems for instance? Using a modified version of our SMB, the answer is yes. The SMB comprises two cell types, one being the soma, a group of cells that have a longer lifetime than wild type E.coli cells & are unable to replicate. The soma cells have the required characteristics for transient use in open environment.

The next consideration is then: can full differentiation be induced in the SMB? In order to achieve this, two events should be initiated:

  • Induction of a massive differentiation of the germ line into soma cell
  • Followed by selective death of the persistent germ line cells.


For details on proposed technical solutions to this challenge, SEE HERE

Metabolic engineering

A bacterial multicellular organism could allow optimizing the production of compounds deleterious to the cell. If one attempts to optimize the production of such a compound in a classical bacteria (E.coli for instance), trade-off appears between the production of your compound and the growth of the cell. The synthetic multicellular organism could in part bypass this problem by partially decoupling growth from synthesis of the exogenous compound. It could indeed be modified so that only the soma produces the noxious molecule. If a mean exists to screen for maximum production of this molecule, you could then select the germline whose soma would have the best exocompound production yield. As long as the production doesn't impair too much the capacity of the soma to feed the germline, the optimization can go on. Thus, a trade-off also exists in this case, but it would very well less stringent that in the wild type E.coli case.

Of course, all this is only possible if the germ line is not affected by the products released by the soma. Compounds that could be optimized in this way are thus constrained to molecules that can be noxious if accumulated in the cell, but that do not affect it too much if in the medium. For instance, if the product can be gradually filtered out of the medium.

E. Colight: towards a new slim diet

Project

In an attempt to integrate all the proposed applications of the SMB, we advanced towards the construction of Ecolight. Bringing together the synthetic organism (SMB), noxious compound synthesis ("metabolic plant"), and the security device. This would include optimizing the production of triglycerides in the soma cells of our synthetic multicellular bacterium. Soma isolation according to the "security device" would be included. The super triglycerides-producing-not-able-to-divide soma cells could then be ingested. The fatty acids they would store would be as many fatty acids you will not absorb! Eat fat, don't get fat!

E.colight project is also made up with 2 independent parts. First we have to create the SMB to implement the secured module. Second E.coli has to absorb free fatty acid and to synthesize triglycerides in the cytoplasm.


Securitydevice4.jpg


References:
- 2006 Physicians' Desk Reference (PDR). Thomson PDR
- BBa_I718002


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