Team:Tokyo Tech/Project/wolf coli


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iGEM Tokyo Tech 2010 "E.coli with Humanity"

4 Wolf coli. Overview



Have you heard the legend of 'The Wolfman'? They're ordinary man at daytime, but suddenly transform into a ferocious wolf in the full-moon night. Our project aim to imitate the character of Wolfman, more specifically, designing two types of E.coli that helps each other to survive at daytime, whereas competing at full moon night.

Figure3-1. Introduction of “Wolf coli.”

Figure3-2. Overview of “Wolfcoli” system

In order to create the “Wolf coli.”, we introduced " red-light-dependent gene expression network"[1] and "band-detect network"[2] and combined these networks with the Artificial Cooperation System (Fig. 3-2). We conducted experiment on series of OmpC promoter (PompC), and LacIM1 which are crucial parts of our networks.

Figure3-3. The induction of new PompC series in high osmolarity medium at hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA
Figure3-4.Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko ODERA

We succeeded in designing 2 new osmoregulative promoters, PompC(CB) (BBa_K395302) and PompC(CS1) (BBa_K395303), which can be utilized in the red-light-dependent gene expression network (Fig. 3-3).

We characterized lacIM1 (BBa_K082026), a mutation of LacI (BBa_?????),which is a key component in the band-detect network. In fact, this part was registered by USTC(2008) [3], however it was not well characterized in the BioBrick registry. We found that product of lacIM1 shows weaker repression to Plac than its wild type. (Fig. 3-4)

Wolf coli. and Artificial Cooperative System

Tokyotech Cooperative activity.jpg

The Aftificial Cooperation System was designed to be switching off during the “full moon night” which means sympathetic coli. would be transformed into wolf coli. by induction of the “full moon light”. During this period, communication between 2 types of cells is inhibited. Therefore, both types of cells are competitors and they are unable to recognize each other by quorum sensing.

Artificial Cooperation system turns on during the daytime and the crescent-moon night which means the systems is activated by either weak or strong light intensity. In contrast, the system is off during the full moon night due the inhibition of intermediate light intensity.

POmpC in red-light-dependent gene expression network

Figure3-2. Overview of “Wolfcoli” system PompC(WT) and the two-component system

OmpC promoter, in red-light-dependent gene expression network, plays a crucial role in initiating the transformation of sympathetic coli. into Werewolf coli. in light sensitive system. To accomplish band-detect circuit in light sensitive system, varieties of OmpC promoters were designed and characterized in order to find an appropriate strength of the promoter particularly which can be activated by the “full moon light”.

LacIMI in band-detect network

LacIM1 is a crucial component in the band-detect network (Fig. 3-2).