Team:ETHZ Basel/Project/Movie

From 2010.igem.org

(Difference between revisions)
(→Project Overview)
Line 3: Line 3:
= Project Overview =
= Project Overview =
-
=== E. lemming ===
+
E. lemming, aims to modify the chemotaxis property of E.coli such that, instead of response to a chemical attractant or repellent, the bacterium responds to a light stimulus. Furthermore, this light sensitivity is used to control E.coli’s movement by deciding, at any given time, which type of motion the bacterium will adopt (tumbling or straight run). This leads to a controllable E.coli, which can move to a defined direction, as a result of the combination of tumbling and straight run. The bacteria in the experiment are imaged and, by image processing, the position of a single tracked cell is inferred. By activating a light switch, the user decides whether the bacterium should continue running or should change direction.
-
[[Image:Setup.jpg|thumb|400px|'''Figure 1.''' Setup to control ''E. coli'' movements. An automatized microscope images the E. lemming. A connected computer system detects and tracks the cells. The direction of movement of the E. lemming is compared to the desired direction defined by the user, e.g. with a joystick. If the direction of movement deviates too much from the desired direction, the digital controller induces tumbling by sending a red light pulse. Otherwise, tumbling is repressed by sending a far-red light pulse.]]
+
-
The core idea of our project is to control chemotaxis of ''E. coli''-
+
== The network topology==
-
by means of light! We'll realize this by hijacking and perturbing the tumbling / directed flagellar movement apparatus. By coupling directed flagellar movement regulating proteins to a '''novel synthetic light-sensitive spatial localization system''', their activity can be controlled reversibly. A light-sensitive dimerizing complex fused to this regulating proteins at a spatially fixed location is induced by light pulses and therefore localization of the two molecules can be manipulated.  Tumbling / directed flagellar movement rates are monitored by image processing algorithms, which are linked to the light-pulse generator. This means that ''E. coli'' tumbling is induced or suppressed simply by pressing a light switch! This synthetic network enables control of single E. coli cells: '''We'll make them move like mindless "Lemmings"''' in the direction they are forced to go!
+
Many bacteria posses a signal transduction network that makes it possible to sense the changes in concentration of a certain chemical attractant/repellent in the extracellular environment and direct the movement towards the attractant and away from the repellent. This property is what is known as ‘''chemotaxis''’. There are basically two types of movement that the bacterium can employ: ''''tumbling'''', which means change of direction and occurs when the bacterium doesn’t sense an increase of attractant concentration in the extracellular environment anymore and ''''running straight'''', which occurs when the attractant concentration is increasing. The running straight stands for “correct direction, ok, keep going” (towards the attractant/away from repellent) while the tumbling stands for “wrong direction, give it another shot”. In the case of E.coli, these two types of movements correspond to different rotation directions of its 4 flagellar motors (clockwise: tumbling; counter-clockwise: runs).
-
<!--- Old version
+
The direction of rotation of the motors can be determined by the ratio of concentrations of certain proteins inside the cell. That is, there are some quantitative indicators from which one can infer whether the bacterium will run or it will go straight, as a response to changes in input concentration. These indicators (concentration levels for certain proteins) are the result of a well – studied signal transduction network, consisting of membrane receptor proteins and intracellular proteins (Che).
-
This years ETHZ Basel project goal is to control  This system enables to control single E. coli cells to move like mindless "Lemmings" in the direction they are forced to go.
+
 
-
--->
+
CheW and CheA are localized right next to the membrane, inside the cell. They form a complex together with the receptor clusters on the membrane (which sense the changes in input concentration in the extracellular environment). This complex can be successively methylated (on different methylation states), a constant process done by CheR. In the literature, there are usually 5 methylation sites considered (m = 0,1,2,3,4; m=0 stands for no methylation and m=4 stands for the highest methylation level). <br>
 +
The key process that influences the outcome of the network (tumbling/running) is the autophosphorylation of the protein CheA, which is favoured either by the increase in the methylation state of the membrane complex, done by CheR, or by decreasing attractant concentration.
 +
 
 +
With decreased attractant (increased repellent) concentration, CheA is autophosphorylated. The phosphoryl groups are being used to produce either CheYp or CheBp. CheYp diffuses through the cytoplasm to the motors, generating clockwise spins, therefore tumbling. CheBp is responsible for de-methylation of the receptors, which reduces the chances of CheA being autophosphorylated, therefore the system is returning to its previous state.
 +
 
 +
With increased attractant (decreased repellent) concentration, the rate of CheA autophosphorylation is reduced, meaning that there are less phosphate groups available for CheYp and CheBp. Therefore the level of CheYp decreases and the level of CheY increases, which leads to straight runs. Then, the level of CheBp decreases, increasing the chances of CheA being autophosphorylated (since the receptors are methylated by CheR), therefore returning the system again to its previous state.

Revision as of 11:06, 22 September 2010

Project Overview

E. lemming, aims to modify the chemotaxis property of E.coli such that, instead of response to a chemical attractant or repellent, the bacterium responds to a light stimulus. Furthermore, this light sensitivity is used to control E.coli’s movement by deciding, at any given time, which type of motion the bacterium will adopt (tumbling or straight run). This leads to a controllable E.coli, which can move to a defined direction, as a result of the combination of tumbling and straight run. The bacteria in the experiment are imaged and, by image processing, the position of a single tracked cell is inferred. By activating a light switch, the user decides whether the bacterium should continue running or should change direction.

The network topology

Many bacteria posses a signal transduction network that makes it possible to sense the changes in concentration of a certain chemical attractant/repellent in the extracellular environment and direct the movement towards the attractant and away from the repellent. This property is what is known as ‘chemotaxis’. There are basically two types of movement that the bacterium can employ: 'tumbling', which means change of direction and occurs when the bacterium doesn’t sense an increase of attractant concentration in the extracellular environment anymore and 'running straight', which occurs when the attractant concentration is increasing. The running straight stands for “correct direction, ok, keep going” (towards the attractant/away from repellent) while the tumbling stands for “wrong direction, give it another shot”. In the case of E.coli, these two types of movements correspond to different rotation directions of its 4 flagellar motors (clockwise: tumbling; counter-clockwise: runs).

The direction of rotation of the motors can be determined by the ratio of concentrations of certain proteins inside the cell. That is, there are some quantitative indicators from which one can infer whether the bacterium will run or it will go straight, as a response to changes in input concentration. These indicators (concentration levels for certain proteins) are the result of a well – studied signal transduction network, consisting of membrane receptor proteins and intracellular proteins (Che).

CheW and CheA are localized right next to the membrane, inside the cell. They form a complex together with the receptor clusters on the membrane (which sense the changes in input concentration in the extracellular environment). This complex can be successively methylated (on different methylation states), a constant process done by CheR. In the literature, there are usually 5 methylation sites considered (m = 0,1,2,3,4; m=0 stands for no methylation and m=4 stands for the highest methylation level).
The key process that influences the outcome of the network (tumbling/running) is the autophosphorylation of the protein CheA, which is favoured either by the increase in the methylation state of the membrane complex, done by CheR, or by decreasing attractant concentration.

With decreased attractant (increased repellent) concentration, CheA is autophosphorylated. The phosphoryl groups are being used to produce either CheYp or CheBp. CheYp diffuses through the cytoplasm to the motors, generating clockwise spins, therefore tumbling. CheBp is responsible for de-methylation of the receptors, which reduces the chances of CheA being autophosphorylated, therefore the system is returning to its previous state.

With increased attractant (decreased repellent) concentration, the rate of CheA autophosphorylation is reduced, meaning that there are less phosphate groups available for CheYp and CheBp. Therefore the level of CheYp decreases and the level of CheY increases, which leads to straight runs. Then, the level of CheBp decreases, increasing the chances of CheA being autophosphorylated (since the receptors are methylated by CheR), therefore returning the system again to its previous state.

Retrieved from "http://2010.igem.org/Team:ETHZ_Basel/Project/Movie"