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.Affinity Tag]] | [[Chiba/Communication|2.Communication Module]] | [[Chiba/Quorum_Sensing|3.Size Control]] ) |  [[Chiba/Making Marimo|Making Marimos]] |  [[Chiba/Goal|Our Goal]]<br>
+[[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]]
 |}
 ==Making Marimos==
-[[Image:final.gif|frame|center|'''Fig1.''' Scheme of final gene cuircuit for Bacteria Marimo.]]<br>
+[[Image:final.gif|frame|center|'''Fig. 26''' Scheme of final gene cuircuit for Bacteria Marimo.]]<br>
 ==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.
-===[[Chiba/Flagella/FliC-his_generator|FliC-His generator]]===
+===[[Chiba/Flagella/FliC-his_generator|Moving FliC-His generator]]===
 ====Experiment====
-*We regulate His-tagged FliC by lux promoter. Namely, if LuxR is expressed, bacteria can express FliC by Quorum Seinsing.<br>
+[[Image:FliC-His ligation.jpg|frame|'''Fig. 27''' Moving FliC-generators into p15A plasmid (not biobricked yet!)]]
-*Because the gene circuit of Quorum Sensing is on vector of colEI, we can use FliC and Quorum Sensing by using vector of p15A and do double transformation.
+*We regulate the expression of His-tagged FliC by lux promoter. Namely, if LuxR is expressed, bacteria can express FliC ''via'' Quorum Seinsing.<br>
-[[Image:FliC-His ligation.jpg|frame]]
+*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====
-Failed to ligate FliC-His generator and p15A origin vector.<br>
+First attempt to import FliC-His generator into p15A vector (see Fig. 2).<br>
-*混乱しています。ごめん。確認なんですが、FliC-His generatorをp15Aにのせるというのは、Biobrickにするためでないでしょうか。。僕の勘違いかな？。。そもそもQuorum sensing のベクターと、FliC-Hisチェックしてビーズ吸着させたときのベクターとを、ダブルトランスフォーメーションできないんだっけ？byとよたろ
+*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]]===
-====Experiment====
+====Coming Soon====
-*ここの文章の意味が伝わりにくいです。細かい話になりすぎるのであれば、この箇所はカットしてもいいと思います。byとよたろ
+*'''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.
-(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).
-(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.
-(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。)
-====Results====
-Not yet (as of 10/26/2007). Cloning is still underway (ligation failed) -->

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