Team:Paris/Perspectives

Biosynthetic Bottum-up approach

A.DNA nanocar factory

The field of artificial molecular machines and motors is growing at an astonishing rate and is attracting a great deal of interest in nanoscience. Research in the last decade has shown that species made of components like DNA or proteins are attractive candidates. Our aims is to build by a bottom-up approched DNA nanocars that have the properties to produce energy in vitro.

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? </br> Also why in vitro? Do you plan to use our system in cell-free extracts or in living cells?

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.

Moreover,in the FIFO the first in is the first out meaning that the 2-3 genes is still activate cause the one went off ,in our construction we success to bind only the higher part ,to built the lower part we need to express 2-3 gene that will finalize the car structure. This sentence is poorly constructed and full of mistakes

The oscillation of our system can be assimilized to a factory when a certain amount of car his made, we need a little time off to permit them to move ,that will make the place for a new one ,his similar to make cookies when they cook you need to move them to put new ones in the oven. This really is farfetched. What will make the cars moove? from were to were?

Fig.1:biosynthesis by sequential expression of 3 DNA origami

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 broking the hydrogen 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.

What is this red region? How will it allow to use the delivered energy? Plus it's not enough to cite a paper to prove that energy is delivered. If the miRNA is complementary to a region that is already paired, the energy you need to unpair this region is the same that you get when you pair it with the miRNA. Result: 0 energy gain.

In conclusion, these kinds of DNA structures can be suitable, reversible and metastable DNA fuels and a new kind of cargo delivery machine. How will they moves? I doubt that turning their wheels will be of any help...

B.Artificial virus factory

We can also produce a lot of differentes kinds of self-assembly structures like virus for example,HIV:

Fig2: genetic organization of HIV virus

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 Env (without Pol to avoid pathogenicity) the FIFO in this case will be essential.

If you ask us why?

For 3 main reasons :

• First, when they genes 1 and 2 will be expressed the protease P10 will cleave gag that will self-assembled.

• Than when 123 will be expresse Env will get cleaved and self assembled with they products of gag and P10.

• Finaly, when 23 get activate we will increase the quantity of subunits delivered from the clivage of Env.

In conclusion, our sytem will be very useful for the developpement of new drugs against HIV or in new kinds of vectors for 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.

Bibliography

• Folding DNA to create nanoscale shapes and patterns

Paul W. K. Rothemund

• Design of DNA origami

Paul W. K. Rothemund

• An autonomous polymerization motor powered by DNA hybridization.

Suvir Venkataraman, Robert M. Dirks, Paul W. K. Rothemund, Erik Winfree, Niles A. Pierce.

• Catalyzed Relaxation of a Metastable DNA Fuel.

Georg Seelig, Bernard Yurke, Erik Winfree.