From 2007.igem.org

Contents 1 How can we define a multicellular organism ? 2 How to proceed ? 3 What essential gene ? 4 What auxotrophy gene ?

How can we define a multicellular organism ?

There is no universally admitted definition. Wikipedia gives: "an organism (in Greek organon = instrument) is a living complex adaptive system of organs that influence each other in such a way that they function in some way as a stable whole."

For us, the organs will simply be different cells. The definition we retain is that of a multicellular entity with different types of cells fulfilling different complementary tasks.

In most organisms, some cells differentiate to realize a function useful for the organism. By useful, we mean that it will give a better fitness, or in other terms, that it will help producing more offspring. The specialized cells are usually not the same than the ones reproducing the organism, and they loose this ability. Thus, their is a notion of sacrifice of some cells to the profit of those dedicated to reproduction. The cells are able to reproduce the organism represent the germline, the ones which specialize and loose this ability represent the soma.

How to proceed ?

How can we drive a cell differentiate into two distinct lines? The differentiation can either be genetic or epigenetic. In epigenetic differentiation, the two or more lines are distinguished only by different patterns of protein expression, generated by an appropriate biochemical circuit. In genetic differentiation however, individual lines will end up having different genome sequences. As a mean of implementing differentiation, we chose the genetic solution based on a DNA recombination system system. We will introduce a special genetic construct into the bacterial chromosome; in a subset of cells in a population the excision of a genomic cassette will lead to differentiation of these cells into soma. The construct could be simply represented in the following way:

This is the construction of the germline. Here, the essential gene is expressed, but the auxotrophy gene is not. Thus this strain is auxotroph for the metabolite this "auxotrophy gene" produces. If the CRE recombinase is expressed, the essential gene will be exised leading to the following construction:

Here the auxotrophy gene is expressed, but the strain doesn't express the essential gene anymore. It will thus die after a given time. But in the meanwhile, it will hopefully have produced enough of the auxotrophy metabolite to feed the germline.

What essential gene ?

The essential gene must fulfill two main criteria:

• The cells should be able to live as long as possible without it.
• Its deletion should not impair to much the capacity of the cell to produce the auxotrophy metabolite

We choose FtsZ. For more details on our choice, see here.

What auxotrophy gene ?

The auxotrophy gene must fulfill three main criteria:

• The soma cells must be able to overproduce and excrete it
• Their must be a simple auxotrophy to the metabolite
• It would be better if there is a promoter sensitive to the metabolite concentration. If this is the case, we could place the CRE recombinase under the control of this promoter. In this way, the germline will differentiate into soma only if there is a need for the auxotrophy metabolite.

We choose DAP (diaminopimulate). For more details on our choice, see here.