Chiba/Goal

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==Goalのために==
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<html><link rel="stylesheet" href="/igem07/index.php?title=User:Maiko/Chiba.css&action=raw&ctype=text/css" type="text/css" /></html>
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==pLux FliC His-tag==
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[[Image:chiba_logo.png|center]]
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*His-tagを入れたFliCをpLuxの下に置き、LuxRが発現されている条件のもとならば、Quorum SensingでFliCを発現させることができるようにする。
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__NOTOC__
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*Quorum Sensingのための遺伝子回路がcolEI oriのvectorに乗っているために、p15Aのベクターを使いdouble transformation することでQuorum Sensingと合わせることができるようになる。
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{| style="border:0;width:100%;font-family:'Trebuchet MS'" cellpadding="20px" cellspacing="0"
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*pLuxをもつベクター側とFliCをPCRを使って、Ligationさせることで作る。
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| align="center" |
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[[Chiba|Home]]<br>
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<span style="font-size:120%;font-weight:bold;">[[Chiba/Introduction|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]</span><br>
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[[Chiba/Acknowledgements|Acknowledgements]] | [[Chiba/Team_Members|Team Members]] | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | [[Chiba/Members_Only|メンバ連絡簿]]
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|}
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==FliC His-tagのbiobrick化==
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==Our Goal: Yet To Be Done==
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必要なこと
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As Final-Construction, we  will carry out four experiments.
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*puc19 vectorの乗っているのでbiobrickのベクターに乗せる。
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*A test of adsorption between flagella
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*制限酵素サイト(EcoRI,SpeI,PstI)をつぶす。
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*A test to confirm a limit of size
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*A test to form Marimo
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*A test of size control
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*FliC His-TagにはEcoRI,SpeI,PstIが含まれているために、そのままではvectorに入れられない。
 
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#片側をblunt end もう一方をApaIの制限酵素サイトをつけ、PCRする。
 
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#vector側も同様にPCRしLigationさせる。
 
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==AHL依存的にFliCが発現するようになるのか==
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==Marimo をつくる==
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===A test of adsorption between flagella===
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[[Image:Imida2.gif|frame|'''Fig. 28''' A test of adsorption between flagella]]
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====Purpose====
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Confirm adsorption between senders and receivers with His-Tag.
 +
 
 +
====Method====
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#Culture senders and receivers in a test tube.
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#Drop senders into receivers tube and mix cobalt ion. <!-- They start to aggregate because of they have His-Tag. -->
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#<!-- After aggregation, we -->Divide it into two groups. One is added imidazole and the other is non imidazole. Inoculate them on the plates.
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#Check the colony.
 +
=====Prediction=====
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If succeeded, cultures which is added imidazole will form a indipendent colony of senders&receivers. The other (non imidazole) will form colonies of adsorbed senders and receivers.
 +
 
 +
===A test to confirm a limit of size===
 +
 
 +
<br>[[Image:ugen.gif|frame|'''Fig. 29''' A test to confirm a limit of size]]<br>
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====Purpose====
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The purpose of this test is to confirm that Marimo has a limit of size on a plate.
 +
 
 +
====Method====
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 +
 
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#Culture senders and receivers in a test tube.
 +
#Inoculate receivers equally on the plate, and drop senders at the center of it.
 +
#After over night,pick colonies which are near the senders (colonies express GFP), the end of the circle expressed GFP, and out of the green circle.
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#Dilute them.
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#Mix cobalt ion into dilutions and inoculate on the plates.
 +
#Check the colony.
 +
=====Prediction=====
 +
If succeeded,receivers which don’t express FliC (they didn’t sense AHL and express GFP) form colonies one by one. <br>And receivers expressed FliC (they sensed AHL and expressed GFP) form aggregated colonies.)
 +
 
 +
===A test to form Marimo===
 +
 
 +
[[Image:Howto.gif|frame|'''Fig. 30''' A test to form Marimo]]
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====Purpose====
 +
Actually we make Marimo.
 +
*AHL diffuse fast because it is very small molecule.
 +
*Packed senders into a very thin capillary . AHL from it diffuse slowly and form concentrate gradients. AHL gradients enable Marimos to aggregate like a real ball.
 +
 
 +
====Method====
 +
#Culture senders and receivers in each test tube and mix Cobalt ions in receiver's tube.
 +
#Add senders to receivers with a capillary.
 +
#Check Marimos.
 +
=====Prediction=====
 +
If succeeded,AHL from senders spreads.<br>
 +
Receivers express GFP and FliC-His,and besides,bond with cobalt ions.
 +
<br>Finnally,form marimos.<br><br><br><br><br><br><br><br><br>
 +
 
 +
===A test of size control===
 +
[[Image:size.gif|frame|'''Fig. 31''' A test of size control]]
 +
====Purpose====
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Control marimo's size by changing　the ratio of amount of senders and receivers.
 +
 
 +
====Method====
 +
#Culture senders and receivers in each test tube.
 +
#Observe marimo's size by changing the amount of receivers and their types.(const.senders)
 +
#Observe marimo's size by changing the amount of senders.(const.receivers)
 +
 
 +
=====Prediction=====
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Low concentration of senders or  receivers : Mini Marimo<br>
 +
High concentration of senders or receivers : Big Marimo<br><br>
 +
 
 +
==In the Long Run==
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*Make Marimo which cross section is colorful. Receivers form layers and express various fluorescent proteins. When we cut Marimo, We will be able to observe beautiful gradation.
 +
*Bacteria Marimo grows like Real Marimo!
 +
Bacteria Marimo grows when it is lighted as if performing photosynthesis like natural Marimo.
 +
 
 +
'''Light triggered marino growth'''<br>
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[[Image:Light sensing sender.jpg]]
 +
 
 +
This gene circuit contains light sensor gene. LuxI represses light senser.
 +
#Prepare mixture of Senders, Receivers(AHL -> FliC-His and GFP), and Co<sup>2+</sup>. 
 +
#Senders express FliC-His but do not synthesize AHL. Senders aggregate FliC-His and Co ions.
 +
#When the culture is lighted, senders express LuxI and synthesize AHL.
 +
#Receivers express FliC-His and GFP.
 +
#Receivers aggregate around the Senders core.
 +
 
 +
Other than that, we have many FUTURE IDEAS (very far future...far enough we can leave CHIBA University happy) such as Marimo-mediated polous materials/ gels. Also presented are the other ways to make marimo in completely different strategies. To check those out, please visit our [http://chem.tf.chiba-u.jp/igem/ official website] (keep updated; never get frozen)
 +
 
 +
<!--ゲル化した細胞を利用した，バイオクロマトグラフフィ-->
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<!--collect ion ? by tahiro-->

Latest revision as of 06:43, 27 October 2007

Chiba logo.png

Home
Introduction | Project Design ( 1.Affinity Tag | 2.Communication Module | 3.Size Control ) | Making Marimos | Our Goal
Acknowledgements | Team Members | [http://chem.tf.chiba-u.jp/igem/ iGEM Chiba Website] | メンバ連絡簿


Our Goal: Yet To Be Done

As Final-Construction, we will carry out four experiments.

  • A test of adsorption between flagella
  • A test to confirm a limit of size
  • A test to form Marimo
  • A test of size control


A test of adsorption between flagella

Fig. 28 A test of adsorption between flagella

Purpose

Confirm adsorption between senders and receivers with His-Tag.

Method

  1. Culture senders and receivers in a test tube.
  2. Drop senders into receivers tube and mix cobalt ion.
  3. Divide it into two groups. One is added imidazole and the other is non imidazole. Inoculate them on the plates.
  4. Check the colony.
Prediction

If succeeded, cultures which is added imidazole will form a indipendent colony of senders&receivers. The other (non imidazole) will form colonies of adsorbed senders and receivers.

A test to confirm a limit of size


Fig. 29 A test to confirm a limit of size

Purpose

The purpose of this test is to confirm that Marimo has a limit of size on a plate.

Method

  1. Culture senders and receivers in a test tube.
  2. Inoculate receivers equally on the plate, and drop senders at the center of it.
  3. After over night,pick colonies which are near the senders (colonies express GFP), the end of the circle expressed GFP, and out of the green circle.
  4. Dilute them.
  5. Mix cobalt ion into dilutions and inoculate on the plates.
  6. Check the colony.
Prediction

If succeeded,receivers which don’t express FliC (they didn’t sense AHL and express GFP) form colonies one by one.
And receivers expressed FliC (they sensed AHL and expressed GFP) form aggregated colonies.)

A test to form Marimo

Fig. 30 A test to form Marimo

Purpose

Actually we make Marimo.

  • AHL diffuse fast because it is very small molecule.
  • Packed senders into a very thin capillary . AHL from it diffuse slowly and form concentrate gradients. AHL gradients enable Marimos to aggregate like a real ball.

Method

  1. Culture senders and receivers in each test tube and mix Cobalt ions in receiver's tube.
  2. Add senders to receivers with a capillary.
  3. Check Marimos.
Prediction

If succeeded,AHL from senders spreads.
Receivers express GFP and FliC-His,and besides,bond with cobalt ions.
Finnally,form marimos.








A test of size control

Fig. 31 A test of size control

Purpose

Control marimo's size by changing　the ratio of amount of senders and receivers.

Method

  1. Culture senders and receivers in each test tube.
  2. Observe marimo's size by changing the amount of receivers and their types.(const.senders)
  3. Observe marimo's size by changing the amount of senders.(const.receivers)
Prediction

Low concentration of senders or receivers : Mini Marimo
High concentration of senders or receivers : Big Marimo

In the Long Run

  • Make Marimo which cross section is colorful. Receivers form layers and express various fluorescent proteins. When we cut Marimo, We will be able to observe beautiful gradation.
  • Bacteria Marimo grows like Real Marimo!

Bacteria Marimo grows when it is lighted as if performing photosynthesis like natural Marimo.

Light triggered marino growth
Light sensing sender.jpg

This gene circuit contains light sensor gene. LuxI represses light senser.

  1. Prepare mixture of Senders, Receivers(AHL -> FliC-His and GFP), and Co2+.
  2. Senders express FliC-His but do not synthesize AHL. Senders aggregate FliC-His and Co ions.
  3. When the culture is lighted, senders express LuxI and synthesize AHL.
  4. Receivers express FliC-His and GFP.
  5. Receivers aggregate around the Senders core.

Other than that, we have many FUTURE IDEAS (very far future...far enough we can leave CHIBA University happy) such as Marimo-mediated polous materials/ gels. Also presented are the other ways to make marimo in completely different strategies. To check those out, please visit our [http://chem.tf.chiba-u.jp/igem/ official website] (keep updated; never get frozen)


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