Team:Paris/Perspectives

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<font color=red>''Un petit texte pour expliquer la motivation de cette page''  ne serait-ce que tout simplement:
 
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On this page we report some possible uses of the FIFO device in synthetic applications.
 
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General comment: I am not at all against an explorative, speculative section. But it's purpose must be clear very clearly focussed on the interest of the FIFO for putative applications. Discuss each time why the FIFO could be especially interesting for your application. This must be very clear in order for this section to fulfill it's purpose.</font>
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As for the flagella biosynthesis, our FIFO could be useful for many '''bottom-up assembled molecular machines''' that needs to be assembled in a precise order.
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Moreover, as we will show on an example, the FIFO could also be useful in many '''biosynthetic pathways''' subject to competitive alternative pathways and for wich intermediate products must be avoided.
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We will develop below two examples: type III secretion injectisome and PHA biosynthesis pathway.
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== Biosynthetic Bottum-up approach ==
 
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<font color=red> ''C'est un grand titre de section, où le titre d'une section normale qui manque?'' </font>
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== Example 1: type III secretion injectisome ==
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==A.DNA nanocar factory ==
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The injectisome is a complex nanomachine that allows bacteria to deliver protein effector across eucaryotic cellular membranes.
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The injectisome consists of a basal structure, which resembles the basal structure of the flagellum, surmounted by either a needle, a needle and a filament or a long pilus.
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[[Image:injectisome.JPG]]
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The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest  in nanoscience. <font color=red> REFERENCES</font>
 
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Research in the last decade has shown that species made of components like  DNA or proteins are attractive candidates.
 
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<font color=red>FIFO devices could be applied to coordinate sequences of  fabrication or operation steps of nanomachines.  
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The injectisome is related to the flagellum genetically. Studies show that a core of eight proteins share significant similarity with components of the flagellum. From this studies, we can expect that the genes expression is ruled by a sequential FIFO order so we can express it with our bacteriO'clock system .
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Moreover, the injectisome will find a lot of applications for example ,bacteria that express this kinds of structure could :
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* induce apoptosis of Eukaryote cells so we can developed  by our system new kinds of  in-vivo compatible anticancer therapy,
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* be a new way to liberate substances produced by bacteria (see bioplastic application).
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For instance, the bottom-up fabrication of ''DNA nano-cars'' !</font>
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In conclusion, our BacteriO'Clock can be used in a lot of new applications like for instance, bottom-up molecular machines self-assembly or new kinds of optimized chemoreactor.
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Our aims is to build ''DNA nanocars''  by a bottom-up approche!
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==  Example 2: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==
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Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials.
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One illustrative example is polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.
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The biosynthesis of this polymer is currently subject to intensive work.
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It consists in expressing in appropriate quantities 3 enzymes PhaA ,PhaB and PhaC that sequentially process AcetoacetylcoA into its final product PHA.
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This biosynthesis os subjected to 2 constraints :
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* Intermediate products of this pathway are used in alternatives competing metabolic pathways,
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* Only the final product is of interest,so that all intermediates products need to be transformed into PHA.
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<span style="color:blue">You have to explain what you mean by "produce energy in vitro". What energy does it use? to convert into what other energy? or to use in what way? Also, why in vitro? Do you plan to use our system in cell-free extracts or in living cells?</span>
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[[Image:PHA.jpg|center]]
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Our system <font color=red> Which system ? Explain</font>is more efficient than a  classic system because  the FIFO order is very important: the first part will bind by complementarity with part 2 this new part will make the binding site for part 3.  <font color=red> ''Which parts ?  Describe. The figure below is too small''</font>
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Two strategies are commonly used in bioengineering of metabolic pathways :
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* Sequential expression of enzymes involved in the pathways,
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* Or constitutive expressions of all enzymes.
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Because of the above mentioned limitations, none of these approaches are adapted here .
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Using the sequential expression, intermediate products would accumulate and thus be consumed by competing pathways.
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Using the constitutive expression a mixture of final and intermediate product would necessarily be obtained.
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[[Image:car2.jpg|center]]
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Our FIFO could be useful here.
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Indeed a FIFO expression pattern is intermediate between a purely sequential and a purely constitutive expression.
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At some point all enzymes are presents ( no accumulation of intermediate products ) and during the last step only the last enzyme (PhC) is presents ( all intermediate products are consumed).
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Fig.1:biosynthesis by sequential expression of 3 DNA origamis
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Moreover, the fact that our system oscillate could provide to the cell a metabolic recovering phase.
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If we add after purifications of our car  a complementary miRNA to the loop, the competition of the miRNA  will open the DNA strands by broking the hydrogen backbound this processe will delivered energy , the instability of the miRNA may make this processe reversible. <font color=red> ''Please explain more, developing in particular the role of FIFO. To my opinion in this prospective part it does not matter to justify everything, the central point is to present meaningful SCENARIOS for the application of the FIFO''</font>
 
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In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels.
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== Bibliography ==
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<span style="color:blue"> How will they move? I doubt that turning their wheels will be of any help...</span>
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== B.Artificial virus factory ==
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* The type III secretion injectisome  Guy R.Cornelis Nature reviews
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* Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates
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Ilana S Aldor and Jay D Keasling
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Department of Chemical Engineering, 201 Gilman Hall, University of California, Berkeley, 94720-1462, USA
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23 September 2003
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We can also produce a lot of differentes kinds of  self-assembly structures like virus for example,HIV:
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* PHA synthase engineering toward superbiocatalysts for custom-made biopolymers.
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<font color=red> No, you can not produce...  You can say (possibly)...
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Nomura CT, Taguchi S.
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The FIFO device provides also a circuit useful to coordinate the self-assembly of molecular structure. A possible canevas for the production of synthetic virus?</font>
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Department of Chemistry, State University of New York - College of Environmental Science and Forestry, 121 Jahn Laboratory, Syracuse, NY 13210, USA.
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<font color=red> Indeed  a virus such as HIV, displays ans organised sequential activation of genes that could be controled by a FIFO circuit. ''ARE YOU SURE OF THIS''</font>
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[[Image:HIV.jpg|center]]
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Fig2: genetic organization of HIV virus
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HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2), those genes  are be cleaved by  protease P10 and then the subunits of the virus will self-assembled into mature virus. <font color=red>A re-enginered virus could  be based on a FIFO setup where gag p10 is first expressed and Env the last  (without Pol to avoid pathogenicity). In this case
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* when genes 1 and 2 are expressed, the protease P10 cleaves gag that self-assembles.
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* when 123 is expressed,  Env is cleaved and self assembles with the products of gag and P10 that have already been produced.
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* finaly, when 23 is activated alone, this increases the quantity of subunits delivered from the clivage of Env. ''I DO NOT UNDERSTANT''
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''In this setup I maybe understand the sequential order of activation, but I do not see why there should be the same order of inactivation. Justify please''
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</font>
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In our case, we will expressed gag p10 and finaly Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.
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If you ask us why?
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For 3 main reasons :
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* First, when they genes 1 and 2 will be expressed, the protease P10 will cleave gag that will self-assemble.
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* Then, when 123 will be expressed Env will get cleaved and self assemble with the products of gag and P10.
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* Finaly, when 23 are activated alone, we will increase the quantity of subunits delivered from the clivage of Env.
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== Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways ==
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Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.
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Our  study is a new metabolic strategy to generate PHA-hyperproducer strains, that have the properties to be a sequential metabolic pathway, we believe the sequential expression  may increases the  production of purified PHA.
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Our strategy consists on replacing  the RFP,CFP and YFP genes by the PhaA ,PhaB and PhaC genes in our final system (containing oscillation,FIFO,synchronisation modules).
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This strategy is more efficient than a constitutive activation for 3 main reasons:
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*First, in this application the NADPH, which is a cellular metastable  fuel, is used by  the PhaB to  synthesize bioplastic. This molecule is very important for many metabolic pathways in bacteria. A NADPH recuperation step is then needed to ensure other metabolic activities to go on. We can then make the hypothesis that if the PhaB is always  activated the bacteria will get exhausted and die quickly. In our system, the bacteria will have a NADPH recuperation step, this is why we hope bacteria will live more than in an usual chemoreactor.
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*Secondly, we hope  by the order of the FIFO to make a synchronized and sequential expression in order to increase the rate of the PHA biosynthesis.
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*Third, as the quality of the bioplastic increases with time, we could predict the quality of the final  product since we would know the duration of a production cycle thanks to the periodicity of our system.
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<span style="color: blue"> Again, you cannot just state this boldly without constructing an argument. </span>
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<span style="color: blue"> You never explain why your system is better than simply expressing all the genes continuously at a lower level </span>
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<span style="color: blue"> More generally, on this whole page: We don't expect you to give very detailed projects. You just need to give simple ideas for which the interest of the FIFO seems obvious (or at least you need to try making it seem obvious). We are not so much interested in the precise mechanisms or genes involved, but rather in the principles that make your projects interesting.</span>
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[[Image:PHA.jpg|center]]
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== Bibliography ==
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* Folding DNA to create nanoscale shapes and patterns
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Paul W. K. Rothemund
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* Design of DNA origami
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Paul W. K. Rothemund
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* An autonomous polymerization motor powered by DNA hybridization.
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Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.
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* Catalyzed Relaxation of a Metastable DNA Fuel.
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Georg Seelig, Bernard Yurke, Erik Winfree.
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Latest revision as of 04:52, 30 October 2008

Perspectives



As for the flagella biosynthesis, our FIFO could be useful for many bottom-up assembled molecular machines that needs to be assembled in a precise order. Moreover, as we will show on an example, the FIFO could also be useful in many biosynthetic pathways subject to competitive alternative pathways and for wich intermediate products must be avoided. We will develop below two examples: type III secretion injectisome and PHA biosynthesis pathway.


Example 1: type III secretion injectisome

The injectisome is a complex nanomachine that allows bacteria to deliver protein effector across eucaryotic cellular membranes. The injectisome consists of a basal structure, which resembles the basal structure of the flagellum, surmounted by either a needle, a needle and a filament or a long pilus.

Injectisome.JPG


The injectisome is related to the flagellum genetically. Studies show that a core of eight proteins share significant similarity with components of the flagellum. From this studies, we can expect that the genes expression is ruled by a sequential FIFO order so we can express it with our bacteriO'clock system . Moreover, the injectisome will find a lot of applications for example ,bacteria that express this kinds of structure could :

  • induce apoptosis of Eukaryote cells so we can developed by our system new kinds of in-vivo compatible anticancer therapy,
  • be a new way to liberate substances produced by bacteria (see bioplastic application).

In conclusion, our BacteriO'Clock can be used in a lot of new applications like for instance, bottom-up molecular machines self-assembly or new kinds of optimized chemoreactor.

Example 2: metabolic engineering of polyhydroxyalkanoate biosynthesis pathways

Human overpopulation combined with the current lifestyle urges the rational, efficient, and sustainable use of natural resources to produce environmentally friendly plastic materials. One illustrative example is polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions. The biosynthesis of this polymer is currently subject to intensive work. It consists in expressing in appropriate quantities 3 enzymes PhaA ,PhaB and PhaC that sequentially process AcetoacetylcoA into its final product PHA. This biosynthesis os subjected to 2 constraints :

  • Intermediate products of this pathway are used in alternatives competing metabolic pathways,
  • Only the final product is of interest,so that all intermediates products need to be transformed into PHA.
PHA.jpg

Two strategies are commonly used in bioengineering of metabolic pathways :

  • Sequential expression of enzymes involved in the pathways,
  • Or constitutive expressions of all enzymes.

Because of the above mentioned limitations, none of these approaches are adapted here . Using the sequential expression, intermediate products would accumulate and thus be consumed by competing pathways. Using the constitutive expression a mixture of final and intermediate product would necessarily be obtained.

Our FIFO could be useful here. Indeed a FIFO expression pattern is intermediate between a purely sequential and a purely constitutive expression. At some point all enzymes are presents ( no accumulation of intermediate products ) and during the last step only the last enzyme (PhC) is presents ( all intermediate products are consumed).

Moreover, the fact that our system oscillate could provide to the cell a metabolic recovering phase.


Bibliography

  • The type III secretion injectisome Guy R.Cornelis Nature reviews
  • Process design for microbial plastic factories: metabolic engineering of polyhydroxyalkanoates

Ilana S Aldor and Jay D Keasling Department of Chemical Engineering, 201 Gilman Hall, University of California, Berkeley, 94720-1462, USA 23 September 2003

  • PHA synthase engineering toward superbiocatalysts for custom-made biopolymers.

Nomura CT, Taguchi S. Department of Chemistry, State University of New York - College of Environmental Science and Forestry, 121 Jahn Laboratory, Syracuse, NY 13210, USA.

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