Team:Tokyo Tech/Project/wolf coli

4 Wolf coli Overview

Introduction

Figure3-1. Introduction of “Wolf coli.”

Figure3-2. Overview of “Wolfcoli” system

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

Figure3-3. The induction of new PompC series in high osmolarity medium at 4 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 have succeeded in designing 2 new osmoregulative promoters, PompC(CB) [http://partsregistry.org/Part:BBa_K395302 (BBa_K395302)] and PompC(CS1)[http://partsregistry.org/Part:BBa_K395303 (BBa_K395303)], which can be utilized in the red-light-dependent gene expression network (Fig. 3-3). We also characterized LacIM1 [http://partsregistry.org/Part:BBa_K082026 (BBa_K082026)], a mutant of LacIWT,which is a key component in the band-detect network. Although, this part was registered by USTC(2008) [3], it was not well characterized in the BioBrick registry. We confirmed that LacIM1 shows weaker repression to Plac than its wild type. (Fig. 3-4)

Wolf coli. and Artificial Cooperative System

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. Cooperative activity in Artificial Cooperation System is regulated by 3 levels of light intensity which are weak, medium and strong. “Weak light” from the crescent moon enables the Artificial Cooperation System on, thus two types of cell are able to communicate and help each other. “Medium light” during the full moon night can switch the system off resulting in declining of the cooperative activity and appearing of the “Wolf coli”. During the daytime, “strong light” from the sun switches the system on, consequently quorum sensing is activated.

POmpC in red-light-dependent gene expression network

Figure3-2. Overview of “Wolfcoli” system

Figure3-3. The induction of new PompC series in high osmolarity medium at hours. This work is done by Thiprampai THAMAMONGOOD and Taichi NAKAMURA

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 sensening system, varieties of OmpC promoters were designed and characterized so as to find an appropriate strength of the promoter particularly which can be activated by the “full moon light”. see more about OmpC promoter,

LacIMI in band-detect network

Figure3-4.Repression efficiency of LacIM1 (BBa_K395401) / LacIWT (BBa_K395402) exposed to arabinose and IPTG. This work is done by Mitsuhiko ODERA

The band-detect network exhibits transient gene expression in response to concentration of chemical signals. In the band-detect network, LacIM1 is a crucial component due to its repression efficiency can afford the network desired non-monotonic response. According to the assay results, we confirmed that LacIM1 shows weaker repression to Plac than its wild type.(Fig. 3-2). (…see more about lacIM1).