Chiba
From 2007.igem.org
| Line 35: | Line 35: | ||
===[[Chiba/Engeneering_Flagella|Making Affinity Tags]]=== | ===[[Chiba/Engeneering_Flagella|Making Affinity Tags]]=== | ||
| - | [[Image:Chiba_flichisgene.png|frame|'''Fig.''' The short peptide with six histidine (“His-Tag”) was inserted into the fliC D3 domain.]] | + | [[Image:Chiba_flichisgene.png|frame|'''Fig. 3''' The short peptide with six histidine (“His-Tag”) was inserted into the fliC D3 domain.]] |
#Make the affinity tag by inserting the his-tag into the flagellar fillament. | #Make the affinity tag by inserting the his-tag into the flagellar fillament. | ||
#*[[Image:Chiba_check.png]] Sequence confirmed | #*[[Image:Chiba_check.png]] Sequence confirmed | ||
Revision as of 05:04, 27 October 2007
|
Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal |
Project Overview
Why We Make a Marimo : Spherical Multicellular Organism!?
When you see a shape in Nature, you will notice whether a sphere, which is absolutely symmetric in 3D, is really stable or not in Nature. In fact, an oil droplet is a sphere in water. Red blood cell in a hypotonic solution shows its shape change to a spherical balloon. However multicellular organisms have their shape different from a sphere except Marimo. Of course, other algae do not show spherical shape, they live on a surface of stone. It is quite intriguing how Marimo remains its spherical shape in a lake!
Our team focuses understanding how such spherical structure can be sustained even when it Is multicellular organisms.
more...
How To Make Our Marimo
- Affinity Tag.
- Communication Module.
- Size Control.
Two cells are used in our system: AHL senders and receivers. Senders generates the affinity tags constitutively, while receivers generates them only when they are induced by AHL. The marimo-forming goes like this:
- Make the sender core by sticking with the affinity tag.
- Insert the sender core into the receiver culture.
- The sender core produces AHL, which make the near receivers to generate the affinity tags and GFPs.
- The affinity tagged receiver sticks with the central sender core. This will continue until the AHL cannnot reach the marimo boundary,
- When the AHL reached the marimo boundary, the adsorping stops, which makes a finite-sized marimo bacteria.
Experiments Overview
Making Affinity Tags
- Make the affinity tag by inserting the his-tag into the flagellar fillament.
Sequence confirmed
- Swarm confirmed
- Flagella strained with anti-flagella antibody
- Phenotype confirmed
- Affnity confirmed
Making Communication Module
- Making Receivers
- AHL > GFP generator (constitutive aiiA) : BBa_T729006
- AHL > GFP & aiiA generator : BBa_I729005
- Sensitive AHL > GFP generator : BBa_I729004
inverter-aiiA
- Making Senders
- MetK Sender : Could not deposit to registry
Controlling Size
- Improve Sender
- Overexpressed the metK (the AHL precursur synthesis enzyme) in the hope to increase AHL synthesis. : Turned out not to work
- Improve Receiver
- Inserted 2 mutations in LuxR which is known from the paper to increase activity.
- Localize AHL
- Tested the GFP expression in constitutive aiiA generator receiver : Went too much.
- Tested the GFP expression in AHL-induced aiiA generator receiver : Needed to twist the circuit more.
- AHL-induced inverter aiiA receiver. Not yet assembled.
Making Marimo
- Moving FliC-His generator. : Not yet assembled.
- FliC-his biobrick : Not yet assembled.
Our Goal
Below we describe the goal to make a marimo bacteria.
- A test of adsorption between flagella
- A test to confirm a limit of size
- A test to form Marimo
- A test of size control
Advanced Goal in Our Future
See below link: our brainstorming of the marimo future!
