Team:UC Chile/Description




Whateversisome: A new platform for in vitro metabolic engineering

We are working towards reengineering the carboxysome, a bacterial microcompartment (BMC), to produce a genetically encoded platform for optimized packaging of metabolic processes. We are introducing recombinant enzymes inside the carboxysome: as a separated space, this compartment is increasing the encounter probability between the substrate and the enzyme, which maximizes the generation of the product. A bacterium having this synthetic organelle would maximize the production of anything needed.

Then, the next step is to get rid of the bacteria. If science has been able to construct a 96-well plate, just like the distribution kit from iGEM HQ, wouldn’t be amazing to have your own kit with recombinant carboxysomes, ready to create any metabolic pathway there is or that you can imagine? The combinatorial possibilities are vast. Any enzymatic route would only take the necessary steps to get there: no problems such as low yield because your product is being used somewhere else, no sequestration, no need for a bypass and no toxicity in the cell. You would only need to combine the whateversisomes that you want, add the reagents and co-factors and wait for your product.

We, team UC Chile 2013, are trying to bring that possibility closer to Synthetic Biologists around the world. In our project, we worked towards creating this world of in vitro reactions inside the Carboxysome. However, first this BMC needs to be reengineered to become a Synthetic Biology tool.

With this purpose on mind, we tried to answer the following questions:

1) Formation:

How does the expression of this microcompartment in E. coli works? How do we obtain an assembled BMC? Can we regulate the induction of the Carboxysome’s plasmid?

2) Targeting:

How do RuBisCO and the Carbonic Anhydrase goes inside the Carboxysome? Is there any sequence that can target any protein of interest to the inside of the microcompartment?

3) Purification and isolation:

How can we purify, in an easy way and without the need of any special equipment, assembled and functional carboxysomes?

4) Disruption and stability:

Can we design a circuit to lyse cells and after that obtain the products stored inside the BMC? How can we use an autolysis to improve purification techniques? How long is our isolated BMC going to last? What are the best conditions to keeping it assembled?

5) In vitro reactions:

Can Carboxysomes do step by step in vitro reactions? Can one Whateversisome use a substrate, convert it into a product, and then another one use that product and transform it into something else?

For more information about each of them, we invite you to explore our wiki. Enjoy!