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Team:iHKU/Project
From 2008.igem.org
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Overview
Pattern formation is one of the most common yet fascinating biological phenomena happening in our daily lives, though for centuries, biologists, physicists and mathematicians have struggled to understand its nature. How do living cells form highly ordered patterns, without a leader or a exotic command? How can our hands, our eyes, our bones form their shape with such an extremely low mistake rate? This question is fascinating but crucial. Our group is now focusing on this issue. The aim of our project is to control a simple pattern formation → ring formation based on a strain of E. Coli that we created, meanwhile by the conditions used in controlling the patterns, to find out an elucidation of the pathways in pattern formation.
As we know, bacteria use their flagella to move around. To reach a recognizable and stable pattern, we must control this random movement. This can be accomplished by quorum sensing and modification of the related genes. There are several key genes responsible for the movement of flagella, two of them are cheY and cheZ. cheY protein has two forms: its phosphorylated form makes flagella rotate clockwise and the cell will tumble; its dephosphorylated form makes flagella rotate counterclockwise and the cell will be driven straight in a direction (run). cheZ protein can help the progress of dephosphorylation of protein cheY. So by controlling the expression of cheZ we can control the cell movement. MG3 is a strain in which gene cheZ is knocked out from its genome. Plasmids containing gene cheZ with selected promoters will response to the quorum sensing signals, and gene cheZ is expressed or inhibited consequently, and the cell will “run” or stay in one place. Thus by controlling the initial conditions, quorum sensing between cells will lead them to form different patterns.
Project Details
- In the beginning we managed to hold control on the motility of the cell. By knocking out the cheZ, a gene responsible for the regulation of cell motility, we generated a cell strain liable for motility regulation—MG3.
- In order to hold a control on the motility of the uniform cells according to the location of the cell, we design the genetic circuit with the cheZ at the downstream of the quorum sensing system.
- We have been synthesized the circuit part by part. Firstly the quorum sensing part of luxR, luxRI2 and plux,then the cheZ part. After testing and calibrating the two part seperately by reporter gene assay and migration test, we combine the two parts in MG3.
- The transformed MG3 was applied to the LB Agar medium to observe the pattern formed. So far we have already achieved different forms of concentric cycles on the Agar plate. And we are now to have finer regulation on the pattern formed by varifying the level of gene expression and the enviromental factors. And in the end we will come to a math model and hold complete control on the pattern by varifying the parameters.
