Team:ETHZ Basel/Biology

From 2010.igem.org

(Difference between revisions)
Line 3: Line 3:
<br>Currently we are working on putting all the BioBricks into a storage vector. The image [[Image:Constructs.jpg|200px|thumb|right|fusion proteins]] shows all the constructs we plan to clone.
<br>Currently we are working on putting all the BioBricks into a storage vector. The image [[Image:Constructs.jpg|200px|thumb|right|fusion proteins]] shows all the constructs we plan to clone.
 +
 +
== Overview ==
 +
 +
The goal of our iGEM project E.lemming is to take over the control of the tumbling frequency of E.coli. This is achieved by reversibly localizing certain elements of the chemotactic pathway (che-proteins) and thus decreasing their activity.
 +
For localization we use the PhyB-PIF3 system found in plants. PhyB or PIF3 is fused to a che-protein while the other one is fused to a localized protein (localizer) within the cell. Upon a light stimulus of a certain wavelength PhyB is activated, binds PIF3 and like this localizes the che-protein. A light stimulus of a different wavelength reverses this process.
 +
In order to increase the probability that the system will work we will try out a lot of combinations of several proteins:
 +
*che-proteins: cheY, cheB and cheR
 +
*localizer: trigger factor (binds the ribosome), MreB (actin anologue), tetR (binds tetO)
 +
 +
== Cloning strategy ==
 +
 +
As we plan to generate several fusion proteins with different linkers, we decided to use the cloning strategy BBF RFC 28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI (http://dspace.mit.edu/handle/1721.1/46721).
 +
The advantage of this strategy is that we can clone up to 3 different inserts into one vector simultaneously and this in a 96 well format.
 +
 +
== Parts ==
 +
 +
<br>Currently we are working on putting all the BioBricks into a storage vector. The image [[Image:Constructs.jpg|200px|thumb|right|fusion proteins]] shows all the constructs we plan to clone.
 +
 +
== working process ==
 +
=== generation of parts ===
 +
==== generation of storage vectors ====
 +
The working process for the generation of the storage vectors is as follows:
 +
Ordering of primers (if template is available) -> PCR -> clean-up of PCR product -> ligation into storage vector -> transformation of competent cells -> plating of cells -> selection of clones (blue-white-screening) -> sequencing
 +
For proteins for which no template is available we let the let the genes synthesize directly.
 +
{| border="1"
 +
|+ subparts
 +
! Protein !! terminus on fusion !! status
 +
|-
 +
! Row heading 1
 +
| CheY || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| CheY || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| CheR || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| CheR || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| CheB || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| CheB || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| TrigF || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| MreB || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| MreB || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| Cell 2 || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| Cell 2 || Cell 3 || Cell 3
 +
|-
 +
! Row heading A
 +
| Cell 2 || Cell 3 || Cell 3
 +
|}

Revision as of 08:40, 29 July 2010

As we plan to generate several fusion proteins with different linkers, we decided to use the cloning strategy BBF RFC 28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI (http://dspace.mit.edu/handle/1721.1/46721).


Currently we are working on putting all the BioBricks into a storage vector. The image
fusion proteins
shows all the constructs we plan to clone.

Overview

The goal of our iGEM project E.lemming is to take over the control of the tumbling frequency of E.coli. This is achieved by reversibly localizing certain elements of the chemotactic pathway (che-proteins) and thus decreasing their activity. For localization we use the PhyB-PIF3 system found in plants. PhyB or PIF3 is fused to a che-protein while the other one is fused to a localized protein (localizer) within the cell. Upon a light stimulus of a certain wavelength PhyB is activated, binds PIF3 and like this localizes the che-protein. A light stimulus of a different wavelength reverses this process. In order to increase the probability that the system will work we will try out a lot of combinations of several proteins:

  • che-proteins: cheY, cheB and cheR
  • localizer: trigger factor (binds the ribosome), MreB (actin anologue), tetR (binds tetO)

Cloning strategy

As we plan to generate several fusion proteins with different linkers, we decided to use the cloning strategy BBF RFC 28: A method for combinatorial multi-part assembly based on the Type IIs restriction enzyme AarI (http://dspace.mit.edu/handle/1721.1/46721). The advantage of this strategy is that we can clone up to 3 different inserts into one vector simultaneously and this in a 96 well format.

Parts


Currently we are working on putting all the BioBricks into a storage vector. The image
fusion proteins
shows all the constructs we plan to clone.

working process

generation of parts

generation of storage vectors

The working process for the generation of the storage vectors is as follows: Ordering of primers (if template is available) -> PCR -> clean-up of PCR product -> ligation into storage vector -> transformation of competent cells -> plating of cells -> selection of clones (blue-white-screening) -> sequencing For proteins for which no template is available we let the let the genes synthesize directly.

subparts
Protein terminus on fusion status
Row heading 1 CheY Cell 3 Cell 3
Row heading A CheY Cell 3 Cell 3
Row heading A CheR Cell 3 Cell 3
Row heading A CheR Cell 3 Cell 3
Row heading A CheB Cell 3 Cell 3
Row heading A CheB Cell 3 Cell 3
Row heading A TrigF Cell 3 Cell 3
Row heading A MreB Cell 3 Cell 3
Row heading A MreB Cell 3 Cell 3
Row heading A Cell 2 Cell 3 Cell 3
Row heading A Cell 2 Cell 3 Cell 3
Row heading A Cell 2 Cell 3 Cell 3
Retrieved from "http://2010.igem.org/Team:ETHZ_Basel/Biology"