Team:Paris/Perspectives

From 2008.igem.org

(Difference between revisions)
(DNA nanocar factory)
 
(71 intermediate revisions not shown)
Line 1: Line 1:
{{Paris/Menu}}
{{Paris/Menu}}
 +
{{Paris/Header|Perspectives}}
-
== Biosynthetic Bottum-up approach ==
 
-
==A.DNA nanocar factory ==
+
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.
-
The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest  in nanoscience.
+
== Example 1: type III secretion injectisome ==
-
Research in the last decade has shown that species made of components like  DNA or proteins are more attractive candidate.
+
-
Our aims is to build by a bottom-up approched  DNA nanocars that have the properties to  produce energy in vitro.
+
-
Our system 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.
+
-
[[Image:car2.jpg|center]]
+
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.
-
Fig.1:biosynthesis by sequential expression of 3 DNA origami
+
[[Image:injectisome.JPG]]
-
If we add in vitro a complementary miRNA to the red regions (fig.2), the competition of the miRNA  will open the DNA strands by broken the hdrogen backbound this processes  deliveres  minimun ten time more energy than  ATP biodegradation (winfree et.al), the instability of the miRNA may make this processe reversible.
 
-
In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine.
 
-
== Artificial virus factory ==
+
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.
-
We can also produce a lot of differentes kinds of  self-assembly structures like virus:
+
==  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.
-
== HIV ==
+
[[Image:PHA.jpg|center]]
 +
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.
-
[[Image:HIV.jpg|center]]
+
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).
-
Fig2: genetic organization of HIV virus
+
Moreover, the fact that our system oscillate could provide to the cell a metabolic recovering phase.
-
HIV can be produce by sequential expression of 3 genes gag,env and pol (Fig.2)those genes  will be cleaved by  protease P10 and than the subunits of the virus will self-assembled into mature virus.
 
-
In our case, we will expressed gag p10 and finaly Pol (without Pol to avoid pathogenicity) the FIFO in this case will be essential.
 
-
If you ask us why?
+
== Bibliography ==
-
For 3 main reasons :
+
* The type III secretion injectisome  Guy R.Cornelis Nature reviews
-
First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag  that will self-assembled.
+
* 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
-
Than when 123 will be expresse Pol will get cleaved and self assembled with they products of gag and P10.
+
* PHA synthase engineering toward superbiocatalysts for custom-made biopolymers.
-
 
+
Nomura CT, Taguchi S.
-
Finaly, when 23 get activate we will increase the quantity of subunits delivered  from the clivage of Pol.
+
Department of Chemistry, State University of New York - College of Environmental Science and Forestry, 121 Jahn Laboratory, Syracuse, NY 13210, USA.
-
 
+
-
In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kind of vectors in genetic therapy.
+
-
 
+
-
== 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 such as polyhydroxyalkanoic acids (PHAs), whose production/degradation cycle reduces undesirable wastes and emissions.
+
-
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.
+
-
 
+
-
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).
+
-
 
+
-
This strategy is more efficient than a constitutive activation for 3 main reasons:
+
-
*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 (4-5 days). 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.
+
-
 
+
-
*Secondly, we hope  by the order of the FIFO to make a synchronized and sequential  turn-over of the enzymatic expression in order to increase the rate of the PHA biosynthesis.
+
-
 
+
-
*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.
+
-
 
+
-
[[Image:PHA.jpg|center]]
+
-
 
+
-
== Bibliography ==
+

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.