Chiba/Making Marimo

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Latest revision as of 05:23, 27 October 2007

Making Marimos

Fig. 26 Scheme of final gene cuircuit for Bacteria Marimo.

Parts Construction

Because we found that FliC code includes restriction enzyme(EcoRI,SpeI,PstI) which is used in Biobrick, we divided into plasmid of FliC and one of signal, and aimed the double transformation.

Moving FliC-His generator

Experiment

Fig. 27 Moving FliC-generators into p15A plasmid (not biobricked yet!)

We regulate the expression of His-tagged FliC by lux promoter. Namely, if LuxR is expressed, bacteria can express FliC via Quorum Seinsing.
Because the Quorum Sensing device is on ColE1-type vector, we need to export the FliC unit into the plasmids with compatible origins such as p15A.

Results

First attempt to import FliC-His generator into p15A vector (see Fig. 2).

Communication units are on ColE1 type plasmids. To make this stickly FliC construct compatible with communication circuits, this is an absolute necessity.....
Not yet (as of 10/26/2007). The first ligation sucked. Cloning is still underway.

FliC-his biobrick

Coming Soon

Making Biobrick version of Flic-His and other stickly-FliCs. Unfortunately, Many forbidden sites for Biobrick production throughout the FliC gene. We are now eliminating them one by one by site-directed mutagenesis (ExSite PCR method).

@@ Line 2: / Line 2: @@
 [[Image:chiba_logo.png|center]]
 __NOTOC__
-{| style="border:0;width:100%;" cellpadding="20px" cellspacing="0"
+{| style="border:0;width:100%;font-family:'Trebuchet MS'" cellpadding="20px" cellspacing="0"
 | align="center" |
-[[Chiba|Introduction]] | [[Chiba/Project_Design|Project Design]] ( [[Chiba/Engeneering_Flagella|1.Sticky Hands]] | [[Chiba/Communication|2.Communication]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]] || [[Chiba/Team_Members|Team Members]] | [[Chiba/Members_Only|メンバ連絡簿]]
+[[Chiba|Home]]<br>
+<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>
+[[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]]
 |}
-（このページの存在意義について話し合いたいです古）
-*クオラムとfliC-hisを合体させた実験てやりました？
-*やってないです・・・・
-*flic-his generator & flic-his biobrickは作りましたっけ？
-*ligationがうまくいかず、そこで止まってしまいました。
-*ってことはPCRまではいっているということなら、このページのExperimentとして入れていいんじゃない？by とよたろ
 ==Making Marimos==
-.2.3をくっつけてマリモをつくる！の図．<-Our goal??
+[[Image:final.gif|frame|center|'''Fig. 26''' Scheme of final gene cuircuit for Bacteria Marimo.]]<br>
 ==Parts Construction==
-FliC内にはBiobrickで使用する制限酵素（＿＿）が入っているため，シグナル側のプラスミドとは分けた．<br>
+Because we found that FliC code includes restriction enzyme(EcoRI,SpeI,PstI) which is used in Biobrick, we divided into plasmid of FliC and one of signal, and aimed the double transformation.
-Because FliC have restriction enzyme(EcoRI,SpeI,PstI)used in Biobrick, we divide into plasmid of FliC and one of signal.
+===[[Chiba/Flagella/FliC-his_generator|Moving FliC-His generator]]===
-===[[Chiba/Flagella/FliC-his_generator|FliC-His generator]]===
+====Experiment====
-*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。<br>
+[[Image:FliC-His ligation.jpg|frame|'''Fig. 27''' Moving FliC-generators into p15A plasmid (not biobricked yet!)]]
-We regulate His-tagged FliC by lux promoter. If LuxR is expressed, bacteria can express FliC by Quorum Seinsing.<br>
+*We regulate the expression of His-tagged FliC by lux promoter. Namely, if LuxR is expressed, bacteria can express FliC ''via'' Quorum Seinsing.<br>
-*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。
+*Because the Quorum Sensing device is on ColE1-type vector, we need to export the FliC unit into the plasmids with compatible origins such as p15A.
+====Results====
+First attempt to import FliC-His generator into p15A vector (see Fig. 2).<br>
+*Communication units are on ColE1 type plasmids. To make this ''stickly FliC'' construct compatible with communication circuits, this is an absolute necessity.....<br>
+*Not yet (as of 10/26/2007). The first ligation sucked. Cloning is still underway.<br>
 ===[[Chiba/Flagella/FliC-His_Biobrick|FliC-his biobrick]]===
-What is needed(必要なこと)
+====Coming Soon====
+*'''Making Biobrick version of Flic-His and other stickly-FliCs.''' Unfortunately, Many forbidden sites for Biobrick production throughout the FliC gene. We are now eliminating them one by one by site-directed mutagenesis (ExSite PCR method).
-*Change puc19 vector into biobrick's vector.
-<br>puc19 vectorの乗っているのでbiobrickのベクターに乗せる。
-*Broken restriction sites of enzyme(EcoRI,SpeI,PstI).
-<br>制限酵素サイト(EcoRI,SpeI,PstI)をつぶす。
-*Can't insert vector directly,because FliC-His contains EcoRI,SpeI,PstI.
-#Insert blunt end on one side and ApaI on the other side.
-#Similarly do PCR and ligation in the vector side.
-<br>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。
- #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。
- #vector側も同様にPCRしLigationさせる。
-==Experiments==
-===Stick-or-not Assay===
-====Sample====
-*Plux-fliC
-*Pbad-fliC
-*ここのデータは誰が担当かな？どこまでデータが出せる？byとよたろ
-====Results====
-#Swarm test
-#Stick test

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