Team:ETHZ Basel/Internal

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Internal

This section will be removed before wiki-freeze!

Acknowledged problems/incomplete pages

General remarks

  • Protein names: normally written
  • gene names: italic
  • Directed movement NOT running, please correct!
  • chemotaxis pathway NOT Chemotaxis nor chemotactic
  • E. lemming with space
  • E. lemming vs THE E. lemming: E. lemming: Project name (whole assay), The E. lemming: One single genetically modified E. coli
  • far-red light, not far red light, please correct!
  • internal links - we should refer to other internal pages (hyperlinks) where appropriate, inside the paragraphs.

Suggestions

  • We need to be consistent in the use of formal/informal language. what do you decide upon this?

first paragraph, define 'anchor' and 'tumbling' and 'directed movement' before using the terms
second paragraph: quite unclear, if possible, add more details & explain more
add a few sentences (and link them) regarding ARL & Safety

50 μm (?) high flow channel; remove question mark

in second part of the page, 'cells with highest score'; define the score
clearly define the reference used: [1] Stuurman et al. 2007; could not uniquely identify it

Original sentence 'It should be noticed that this signal changes with time constant in the range of hours' rephrased to:'It should be noticed that this signal constantly changes with time in the range of hours.' Revert if meaning changed.

added reference [2]. would be good if a biologist can check if it's the right one. Looks like the page ends abruptly. something like a conclusion perhaps?

spacing, subheadings & some bullet points); suggesting more images for the user experience part

Corrected minor spelling mistakes. Gave names to the 2 steps of the cell detection algorithm. Added subheadings to make it more readable & structured. Revert if necessary.

check with biology to keep it updated (check list)

Add link to Biozentrum Unibas.

Teaser & Motivation

Images

Simona: Achievements image: increase resolution; Same for first image. (Response from George: resolution was decreased to match to LowRes Lemmings screenshot. It looks incoherent, if only E. lemming is HighRes. See this version.)

Thanuja: a better picture to suggest what we have done? any creative ideas to make a better first impression? (Response from George: Yes, please make suggestions! I ran out of ideas. ;-))

Simona: pictures look very cool; if possible, change the one from 'Information processing' (Response from George: Do you have a suggestion, what we could take?)


Elsa please see my suggestions/feedback in the respective mail (named like this). Text needs improvement, the rest is awesome!


Here my suggestions:

E. lemming- a remotely controlled living robot by ETH Zurich

For the animations: would be nice to have titles with questions, that structure the videos (not yet added in my suggestions).

ANIMATION:

molecular mechanism

the synthetic molecular chemotaxis signal transduction pathway of the ETH E. lemming enables us to switch the flagellar rotary machine between its cw and ccw states, in response to an extracellular input-which is in our case red and far-red light. in wild-type bacteria, chemoattractants and chemorepellants are the stimuli-generating compounds which produce a chemotaxis response: consequently, the prokaryotes, moves away or towards the compound by altering its flagellar tumbling frequency via thenaturally occuring signaling cascade. the E. lemming is addidtionally equipped with a synthetic set of light-inducible proteins and anchor proteins, which add a novel tool to the chemotaxis machinery.

What are the key players in the synthetic cascade and how do they act? The attractant or repellent compound binds to the methylaccepting proteins (MCP), embedded in the (inner) bacterial membrane. Through conformational change, this input signal is conveyed via an elaborate regulatory pathway, including CheW and CheA, to CheY, by tightly adjusting it's level of phosphorylation. CheY in its phoshorylated form can bind to the flagellar motor complex and induce a clockwise rotation of the apparatus, which results in tumbling.

unphosphorylated Chy, in contrast, canot bind to the motor complex. if chey is not phosphorylated, the motor remains rotating in its default ccw direction and the bacterium moves continuously forward. Although constant phases of ccw rotation are time-limited and not long-lasting, the phosphorylation state and frequency of chey has great influence on the tumbling frequency and therefore, chemotaxis as an entire system.

our research therefore aims at manipulating the concntration of free Cheyp available for via synthetic anchor proteins. as phosphorylated chey is then “anchored” to those localizers, the cheyp-concentration is decreased and and the flagellal switch can be inhibited. Via light-sensitive proteins (LSP's), cheyp is reversibly anchored to lsp1, itself bound to lsp2. the “kidnapping” of cheyp this has drastical consequences for its network of downstream partners-and on the tumbling requency of the motor.

When come our light-pulses into play? Well, it's right at this stage! the far-red light induces dissociation of the LSP proteins and Chey is “freed” and released to the cytosol, where it acts as a “tumbling agent” for the prokaryotic motor complex. this results in an increased tumbling frequency.

A pulse of red light induces the fusion of lsp1 and lsp2. again, cheyp is anchored to the lsp1/Lsp2-complex. as a consequence, the motor rotation remains in its default ccw direction and the bacterium moves forward. our research efforts focuse on resolving the questions related to the signaling protein chey

A pulse of red lights removes cheyp from the cytosol(by anchoring it to the lsp1/lsp2-complex) and the bacterium moves forward.

Far-red-light, kidnaps relaeses cheyp to the plasma, as the lsp1/lsp2-complex dissociates and the bacterium tumbles.


A challenging issue was how to automatize the process of assessing the movement of the E. lemming and applying corresponding light pulses. We build an information processing pipeline, consisting of different modules. The imaging module, the controller and the E. lemming itself. The imaging module (consisting of a microscope connected to a controller, detects movement. By comparing the input of the imaging module with the desired direction, the controller determines which light-pulse it has to send. This enables the “user” of the E. lemming to watch its movement on a screen and direct it at the same time via the joystick.

The fusion of all those components into exchangeable standard biobricks, the stimulating interworking between molecular biologists and computer scientists as well as the in vivo and in silico research on the E. lemming brought our bacterial robot alive!



TEASERS:


Motivation and project idea:

E. lemming is the the iGEM research project of ETH Zurich. Our research aims at controling the bacterial chemotaxis network by a toolbox of synthetic light-inducible downstream partners and anchors of CheY, a chemotaxis signaling protein. We furthermore built a computational information processing pipeline, consisting of different modules, interconnected by robust algorithms, cell imaging devicesand-a JOYSTICK able to send various light pulses! Find out how it works and explore our WIKI!


E. lemming-the movie

Watch our introductory movie and get to know the E. lemming, the first genetically engineered living robot in the world-with a remote control!


How is the synthetic signaling network implemented?

The coupling of standard chemotaxis proteins with novel synthetic light-sensitive constructs enables us to reversibly anchor the “tumbling agent” CheY and therefore create a synthetic switch module for the bacterial motor complex. Do you want to find out more? In this section you will be provided with insights into our research on the fusionprotein brickbox, the cloning strategies, the wet lab experiments and much more!



Mathematical modeling

How can modeling be helpful for wet lab research? What is predictive power and what kind of algorithms were used? And what the hell is that joystick good for? blablaetc


Visit our modeling section and see the pictures of our elegant, sophisticated supermodels!


Information processing

nothing to additional


Achievements

We created a BioBrick and a Mathlab Toolkit for our E. lemming robot. ...unique and inspiring interworking process between engineering and molecular biology 8or biochemistry). This spirit was it, what gave the project its heart beat!

modelers could write about their coupling of models, what they achieved by computing and predicting, etc.

biologists: coming soon, I guess. this involves the new parts we generated, etc


rename? --> Meet the team (or: The team)


Who are the crazy inventors of E. lemming? Do they want to take over the world or are they just nice, hard-working students? Meet the team and take a look behind the scenes of ETH's iGEM research project 2010!

Text

Motivation & Project Idea

Simona: E. lemming is the 2010 iGEM project of ETH Zurich! We are controlling the movement of any E. coli cell by hijacking chemotaxis, monitoring its spatial behavior by image processing techniques and directing it towards an user - define target by smart control algorithms! Find out more about our motivation and project idea!

E. lemming - The movie

Simona: Watch a short animation of E. lemming! Find out the core concepts behind our project and how we combined biology, mathematical modeling and image processing techniques to create E. lemming!

Biology & Wet Laboratory

Simona: How is it implemented? By coupling the Che proteins of the chemotaxis pathway to a synthetic light - sensitive spatial localization system, their activity can be reversibly controlled. To find out more about E. lemming on the molecular level and to find information regarding the wet laboratory experiments, visit our Biology & Wet Laboratory section.

Mathematical Modeling

SimonaMathematical Modeling How does modeling help? To find out how the mathematical models we developed not only supported wet lab experiments by optimizing the biological network structure to be used, but also provided a test bench for our information processing system, visit the Mathematical Modeling section!

Information Processing

Simona How is it controlled? Implementation of a comprehensive information processing workflow for controlling E. lemming was achieved by combining microscopy, image analysis and processing and controlling algorithms. Find out more about how the information processing part of our project not completed E. lemming, but also created a brand new synthetic biology game: E.lemming!

Achievements

Simona: OK


Motivation

General

  • Movies: George & Luzi Almost done, but needs speech! Elsa
  • Matlab Toolboxes: Moritz & George

Proofreading

Introduction

Biology & Wet Laboratory

Mathematical Modeling

Christoph will do this by Sunday

Christoph:

  • first box: E. lemming, these aren't the devices we used for modeling, that is what we model, anchor not necessarily the plasmid
  • too many "in order to"
  • mathematical modeling overview: please rearrange: 1. WHAT complex model set up, consisting of chemotaxis, light, move, implemented and coupled 2. WHY explain support a little bit more
  • Model implementation: tab and text order should be equal; implementation and adaption of literature described ones, movement by our own!

C: give an idea about the complexity, e.g. high number of proteins and interactions; why did we use only Spiro and Mello?; models: text first as it explains the plots; please gibe a clearer description of the plots, for an external person it's almost not understandable

Information Processing

Achievements

Pick-a-task

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