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Team:Tokyo Metropolitan/Project/Pattern
From 2010.igem.org
Contents |
E.coli pattern formation
Reaction-Diffusion model
Reaction-diffusion equation proposed by A.Turing, 1952.Turing is the famous mathematician of England, as who invent “Turing machine”. This equation describe reaction and diffusion of two substances, and they form specific pattern autonomously. pattern which read by these substances are similar to animal specific skin pattern such as spot pattern of Panther, stripe pattern of zebra, and so on. But the relationship between equation and animal skin pattern was not clear for long days. in 1995,Kondo.S demonstrated that skin pattern of Pomacanthus can describe by using reaction-diffusion equation. Kondo our purpose is, to demonstrate existence of Turing pattern in organisms, by reproduce patterns using E.coli. and verify the theory.
approach
We described that Reaction-Diffusion equation shows reaction and diffusion of “substances”. To form patterns, these “substances” whould be important. We need morphogen, “Activator” and “Inhibitor” as substance. When these substance fulfill some conditions, patterns would reproduce such as spot patterns like panther, or stripe patterns like zebra and so on. this is already demonstrated in computer simulations.(参考文献) We suppose that a bacteria as an individual cell. These “cells” express the genes which cording patterning substance. and reproduce autonomous patterns by their interact. We have some ideas to make these patterns more interesting. One is use of temperature-sensitive bacteria, to make different patterns by temperature in same culture. Another idea is use of multiple substances. Increasing types of substances, we expect that can make more complicated patterns.
the model
How we meet the condition?
we described that to reproduce the model, we need two substances, Activator and inhibitor. but we need more conditions about relationships between Activator and inhibitor; 1. There are two components- the activator and inhibitor 2.Activator's self activation 3.Inhibitor's activation by activator 4.activator's inhibition by inhibitor 5.diffusion speed: Inhibitor>Activator
by following these condition, we designed the original reaction-diffusion model.
the first condition ”two components- the activator and inhibitor” is already cleared. we designed Activator and Inhibitor like these;
Activator
cAMP(cyclic adenosine monophosphate) is the second messenger of cellular signaling. cAMP activate global transcriptional factor CRP(cyclicAMP receptor protein). in our project, we introduce CyaA(cAMP synthase) to E.coli, and express cAMP. then, activate CRP. this cascade fulfill first condition ,”Self-activation of Activator”. and cAMP secrete from activator cell and effect to Inhibitor cell . so, we can fulfill second condition, ”Activation of Inhibitor by Activator”. We designed bioblick as following. Putting CRP binding site, lac promoter, lacI binding site,(these sites become important to fulfill other conditions. we explain later.) RBS and ompA signal , on the upstream(5’ side) of Bioblick. these parts are Designed by USU iGEM2009,from team Utah state2009, for fused with a silver fusion compatible protein to make a functional device for secretion studies(http://partsregistry.org/Part:BBa_K208017). we use this parts for secretion of cyaA and cAMP. when cellular CRP bind to CRP binding site of this parts, transcription of cyaA, and mRFP1(and terminators) will start. mRFP and teaminators are designed by ~,Misa s. we going to register ~~(parts No.)~~ as a new parts..
Inhibitor
We choose Inhibitor factor as Glucose. when lacZ(βgalactositase/Glucose synthase) express by CRP from Activator cell, synthase transport by ompA signal to periplasm. on periprasm, sythase synthesys Glucose. it effect to Activator cell’s lacI, and repress transcription of Activator plasmid. so, we can fulfill the 4th condition “Activator’s Inhibition by Inhibitor.” Biobrick of Inhibitor have also same structure with Activator. from Promoter to ompA signal using BBa_K208017 too. lacZ parts is taken from BBa_I732901 Designed by Zhan Jian, iGEM07_USTC, which containing full-length of lacZ(http://partsregistry.org/Part:BBa_I732901).
