Team:Osaka/Project

・What is an “Empathetic bacterium”?
What does the term “Empathetic bacteria” refer to? When we talk about empathy, we can say that it is something that could not be generated without a mutual relation between two people. But bacteria are different species from the human being; so to give a description about “Empathetic bacteria” we should define differences between the two species.
An obvious difference between bacteria and us is the fact that bacteria are unicellular and human beings are multicellular organisms. Taking that in account, we thought that, in order to prepare empathetic bacteria, we should produce “multicellular” bacteria first.

・What is a “multicellular bacteria”?
To define any multicellular organism we should start by defining a living being. Then, we can pass to define a multicellular organism.

The definition of a living being is something the human being has been asking himself since ancient times, and is not an easy question to answer at all. For this project we consider a living being has the three following characteristics:

１ Organization: Being structurally composed of one or more cells — the basic units of life. 　
２ Metabolism: Transformation of energy by converting chemicals and energy into cellular components (anabolism) and decomposing organic matter (catabolism). Living things require energy to maintain internal organization (homeostasis) and to produce the other phenomena associated with life. 　
３ Reproduction: The ability to produce new individual organisms, either asexually from a single parent organism, or sexually from two parent organisms.
These complex processes, called physiological functions, have underlying physical and chemical bases, as well as signaling and control mechanisms that are essential to maintaining life.

Given that definition, we define a multicellular organism as a set of cells that do not have the three characteristics above mentioned by themselves, but they have all the characteristics when joined and cooperating for the same objectives as one organism.

・What we prepare in this project
Considering the definitions mentioned above, we tried to make this genetic arrangement.
This circuit is a toggle switch model, with the A and the B-side inhibiting mutually. While in the downstream of the A side there is a gene for a protein expressing fluorescence, in the side B there are genes called ilvE and suIA. ilvE (from isoleucine-valine) is a gene for transaminase B, a enzyme related to the isoleucine byosynthesis. sulA (from suppressor of lon) is a gene which is activated in the SOS response and expresses a protein that inhibits cell division.
Then we introduce this genetic cirucuit in an ilvE delete variant (Cooperative Adaptation to Establishment of a Synthetic Bacterial Mutualism , 2011 K.Hosoda et al ) of E. coli.
As a result, we hypothesized the following result:
１、They carry the same genetic information.
２、Both bacteria express two kinds of phenotypes. 　　
　　→A-side-expressed bacteria: they cannot produce the nutrient (isoleucine), but they can reproduce. As a result, when there is nutrient insufficiency they express the B-side.
　　→B-side-expressed bacteria: they can produce the nutrient (isoleucine), but they cannot reproduce.
３、Bacteria of type A and B live in symbiosis, helping each other. So B-type bacteria offer the nutrient to the A-type ones, and the A-type ones transform into B-type, collaborating with the growing of b-type bacteria population.
　We tried to produce this kind of bacteria and analyze their properties through experiments and simulation.




・Result(Wet)

In this experiment(Wet), we achieved these things. ・We newly standardized E.coli ,ilvE and sulA gene. ・We developed toggle switch construction kit(BBa_K100007~BBa_100013). It helps you when you construct toggle switch circuit. ・To evaluate the function of ilvE, We have constructed this genetic circuit. And we have succeeded in evaluating ilvE function quantitatively. ・Method We have constructed this genetic circle (BBa_K1040013) to assay the function of ilvE gene. We cultured these two kinds of E.coli in M63 medium with the Ile and without the Ile. And, we measured OD every two hours. And we also use BBa_J04450 as a control.









・Result

Ordinarily, E.coli has ilvE gene which encoded a kind of the Ile synthetase. Under the Ile existence, both E.coli having ilvE and E.coli which did not have it increased. When Ile did not exist, there is a clear difference between the growth rate of E.coli having ilvE and E.coli which did not have it. Also, OD under M63 medium (minimum medium) with Ile differed from it under M63 medium without Ile.

・Discussion
Experimental results were almost similar to theory, but OD did not increase very much for several hours after start of measurement. Then, it is thought that Ile necessary for E.coli to increase was short. Therefore, starvation response occurred, and it took time to be replaced by the system that mainly synthesized this amino acid.
Also, we want to make such a sequence and perform biohazard measures. We add Anhydrotetra cyclin to culture medium. Anhydrotetra cyclin inhibits that TetR binds to ptet, so E.coli with this sequence cannot increase out of culture medium.