iGEM – Grand Prize 2010 – Metabolic Engineering and Synthetic Biology: DNA scaffolds for biochemical pathway improvement.

by Bio! Mexico 

Cell signaling pathways and cascades are  classic textbook topics which I discovered in my high school days and which fascinates me ever since, just as it does to any student who (truly) begins his journey into the marvelous realm of Molecular Biology.

Back then, me and my friends -at the Biology Olympiad preparation in Mexico City- use to compete to see who knew best the different kinds of G proteins, the cell responses and the evolutionary conservation of the MAP kinases pathway. I remember very well the morning when one of my friends came to us for breakfast, with the look of someone who spent the whole night reading meticulously, and introduced us to the JAK-STATs with such an enthusiasm that it was midnight when we were done reading and speculating… something that was a sort of a sport for us.

One of those afternoons, I started reading with more detail about a certain kind of proteins: the scaffold proteins. These proteins carry the function of binding to different kinases -or another kind of signaling protein- through their binding domains, so these kinases could be near each other. This spatial localization of interacting proteins facilitates signal transmission. In a word, these scaffold protein bring together interacting signaling proteins to boost the signal transmission.

The years went by, and one day it reached my ears the rumor that an iGEM was being organized in our school –the UANL Biological Sciences School– so we immediately started organizing meetings, in order to generate ideas and a project.

One of these ideas -a product of the oh, so many cups of coffee I drank and the long lonely hours I spent scribbling here and there- consisted on building chimeric enzymes which carried a binding domain, in such a way that when a designed protein scaffold was expressed, those chimeric enzymes would bind to it and come into proximity. If the enzyme used were part of a biochemical pathway, then, this pathway yield would be enhanced.

Among the contributions I claimed the project would make were: the fact that the yield of a biochemical pathway could be enhanced, the possibility of controlling the pathway’s efficiency through the control of the scaffold’s expression, and finally, the possibility of re-routing biochemical pathways through the inclusion of the enzymes responsible of the critical steps.

I cannot describe the surprise I felt when I stumbled upon a publication by Dr. John Dueber, which was entitled: Synthetic protein scaffolds provide modular control over metabolic flux . The authors commented how, using scaffold proteins, they were able to regulate the flux through the mevalonate and the glucaric acid pathways. They presented data about reduction of metabolic strees inside the cells and also pointed that these proceeding could be generalized for other metabolic pathways and that it represented an additional regulation level of protein expression. My ethical sense forced me to present to my team partners the work of Dr. Duebe, along with another interesting data published by Dr. Caleb Bashor.

In spite of my initial enthusiasm and the feeling of having hit with an idea without precedents, I soon noticed that what the idea had of original was the only fact that we would be implementing the scaffold system in BioBrick format. So I started again thinking and wondering -with coffee in hand and my eyes circles in bloom- on how could I improve this scaffold idea… Then, news came from the Massachusetts.

Scheme of a DNA scaffold.
The students from the Ljubljana University had just won the BioBrick Grand Prize after having developed a fabulous solution: instead of using proteinic scaffolds, they designed DNA scaffolds!

Uh, DNA scaffolds, but how do they work? Well, let’s recall from the Cell Biology clasess that the are proteins which have peptidic domains that bind to certain DNA sequences. This DNA-binding domains have been widely used in other applications, like yeast two-hybrid assays, but the Ljubljana 2010 iGEM team had found another extraordinary application: use them to build chimeric enzymes, so that these chimerical construction can bind to DNA in an ordered fashion. In such a way, the enzymes of a pathway could be put near each other just like with protein scaffolds! 

Note that this enzyme recruitment is possible because there is no such thing as a nuclear membrane in bacteria, so DNA is accessible for binding proteins.
Violacein yield as registered when using
an ordered DNA scaffold program (green), 
a scrambled program (red), and a random program 
(purple) as negative control.
Furthermore, the Slovenian team not only made the biosynthesis of violacein and carotenoids more efficient, but they also found that this efficiency increase was more pronounced when the DNA program -i.e. the order of the recognition nucleotide sequence- matched the order of the biochemical pathway. They also explored possible application of DNA scaffolds in the refinement of synthetic genetic circuits, like oscillators.

This huge advanced for Synthetic Biology was presented under the title: «DNA coding beyond triplets» and it is perhaps just a matter of time until we see new results and new applications for this system. And, well… obviously, my iGEM team (UANL_Mexico 2011) then decided to work with something different!

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