Team:Kyoto/CiC/Introduction

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Project2 -- Cells in Cells --

Our project aims to reconstruct mitochondria. Our ultimate goal is synthesizing a cell model. We'll tell you why we got interested in reconstruction of Mitochondria.

Minimalized Genomes?

Inspired by the idea of minimum genomes and synthesizing cells, one of Kyoto iGEM team members came up with the idea of reconstructing mitochondria or chloroplasts, whose genomes have been minimalized in evolutionary history. According to endosymbiotic theory, these organelles originated as bacterial endosymbionts. Thus, reconstructing such organelles, in other words, synthesizing ex-cells, seems to us as interesting as the synthesis of the current bacterial cells. Furthermore, we would like to discuss with scientists who aim to synthesize “naturally occurring cells” from the scratch. If mitochondria or chloroplasts were completely reconstructed by synthetic approaches, what do they think of the idea of “cells”?

What is the Difference between Cells and Mitochondria or Chloroplasts?

Mitochondria and chloroplasts have lost many genes in the evolutionary process of adaptation to environment. Thus, they are strongly dependent on nuclear genome for expressing the essential proteins needed to maintain their functions.This is why they cannot be cultured in medium as E.coli is easily cultured. Many proteins with signal peptides are translated in cytoplasm and imported into mitochondria or chloroplasts through a few translocases on their membranes. Thus, the biological systems in mitochondria do not work without the imported cytoplasmic proteins, although they contain their own genetic systems. In other words, they function and act like living cells only in host cells. In a sense, they can be regarded as “cells in cells” which remain alive only in host cells.

Synthesis of Cells?

We consider reconstructing mitochondria or chloroplasts as primitive cell models. Technologically or financially speaking, a mitochondrion and a chloroplast seem easier to reconstruct than a whole cell because components required for the reconstruction are much less. We mentioned an extreme argument that mitochondria and chloroplasts might be potential cells (in cells) as described above. The reason why we mentioned it is that this project originally had a nuance of irony toward the overwhelming difficulty in and ambiguous definition of synthesizing cells.

What is Interesting in This Project?

Apart from such irony, we believe that reconstruction of mitochondria itself seems interesting and exciting. Though cells are too complicated system, mitochondria and chloroplasts themselves are also complicated enough to tackle. Therefore, one of the keys to solve the problem is the fact that mitochondria and chloroplasts contribute little to maintaining themselves as mentioned above.They cannot manufacture what they need by themselves, so they need to import them (e.g., protein factors) through special loopholes.Imagine what will happen if synthetic vesicles, such as liposomes, with such loopholes (translocases) are put into host cells, and contain full of products of translation. We believe that such "prepared liposomes" could act like mitochondria or chloroplasts by eating and using existing proteins for these organelles.

Mitochondria or Chloroplast?

Based on the above idea, we have designed many types of prepared liposomes, such as one with mitochondrial inner membrane translocases, one with mitochondrial outer membrane translocase, one with chloroplastic inner or outer membrane translocases, each one with its genome and so on.At first we planned to select both mitochondrion and chloroplast as model organisms. Chloroplasts seemed easier to observe because they have more bacteria-like system than mitochondria. And translocases of chloroplasts seemed easier to use because they do not require H+gradient when precursor proteins go across double membranes.But we had difficulty in getting enough information on translocases of chloroplasts.In addition to that, we thought it so difficult to introduce prepared liposomes into plant cells because of cell wall. Thus, we selected mitochondria as model organism, because we thought that mitochondria and mammalian cells for host cells are easier to deal with.

In this “cells in cell“ project, we set three subgoals.

1. Designing and constructing synthetic liposomes that penetrate through human cell membranes.
2. Designing and constructing liposomes which can take in mitochondrial proteins from cytoplasm.
3. Introducing them into host cells.

Reference

Concept of Minumum Genome and Syntesis of Cell

• Scott N Peterson and Claire M Fraser: The complexity of simplicity. Genome Biol. 2001
• Anthony C. Forster and George M. Church: Synthetic biology projects in vitro. Genome Res. 2007
• Anthony C Forster, and George M Church: Towards synthesis of a minimal cell. Molecular Systems Biology (2006)
• Clyde A. Hutchison, III, et al: Global Transposon Mutagenesis and a Minimal Mycoplasma Genome.Science 286, 2165 (1999)

Protein import into chloroplasts

• Paul Jarvis and Colin Robinson: Mechanisms of Protein Import and Routing in Chloroplasts.Current Biology, Vol. 14
• J. Philipp Benz, Jurgen Soll and Bettina Bolter: Protein transport in organelles:The composition,function and regulation of the Tic complex in chloroplast protein import. FEBS Journal 276 (2009)