Tailoring Your Avatar--NYMU iGem 2011 Reference Web Project

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中藥心法

由Wyjbush CHang

釋字627號解釋下的 "總統之刑事豁免權"

由CT Wang

扰乱公共秩序罪

由benno tian

刑诉法的审判管辖

由benno tian

2011 iGem沙龍晚會 (Salon de l'Avatar)

E組帶領分組討論

老師滿希望能著重這部分，回去想想這也是舉辦human practice的原意嘛>///<！

合成生物學與生物倫理

扮演上帝?

強制演化?

神經道德學

身體概念的重塑

人的機械化

各組設計詳細報告

宗教問題

神給予人意志?

訂做你的阿凡達：光磁控遠端神經操作 (Tailoring Your Avatar --Optomagnetogenetic Tele-Neurocontrol)

E組(Panel E) [神經道德學探討、生物倫理學]

當前討論結構_尚叡部份

主軸

長期

中期

短期

在正確的時間.狀況做允許的影響行為

犯罪與濫用

資訊安全

進入迷流狀況不願回到現實? (Inception)

操縱人心?

比例原則

標準操作流程

安全性

理性判斷

F組(Panel F) [贊助廠商與支持來源搜尋]

3. 統計及彙整

2. Proposal寄送及聯繫sponsors

1. 修改Proposal

D組：(Panel D) [網頁呈現、科學視覺化、科學藝術]

Members: 傳胤(組長)、建宏、章樺、嚶蓮、尚叡、顥馨

5. 會議記錄製作

攝影、紀錄

4. 多媒體編輯工具學習

Mediawiki, AI, PS, AutoCAD, Aftereffect, 代工

3. 影片製作

5. 後製

4. 動畫

3. 器材

2. 內容

2. Public Team Wiki製作

3. 與Reference Web Project的結合

2. 架構

1. 風格

1. Schema and concept design confirmation.

C'組(Panel C') [前導實驗與先行分子生物基礎建立]

Magnetospirillum magneticum (AMB-1)向法國吳龍飛教授取得

養菌時間

養菌條件

Nitrogen gas & fermentor

自製培養箱

養氣電極(借用)

氮氣鋼筒(借用)

熱熔膠槍

有力的鐵夾

冷氣用軟管

Lock-Lock最大號保鮮盒

低氧養菌槽(借用)

liquid or solid (with argarose) medium?

MSGM

The medium receipt

Medium Receipt(protocol):

1 liter of magnetic spirillum growth medium (MSGM) contains….

 Distilled water 1.0 L

 10 ml of Wolfe’s vitamin solution

 5 ml of Wolfe’s mineral solution

 2 ml of ferric quinate solution (0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)

 0.68 g of KH2PO4

 0.12 g of sodium nitrate NaNO3

 0.74g of succinic acid

 0.1 g of sodium thioglycolate

＊ Enriched MSGM contains

 0.02% polypeptone

 0.01% yeast extract

 0.005% L-cysteine instead of sodium thioglycolate.



All of the media used were supplemented with 5 mg/ml tetracycline

pH 6.75 with aqueous sodium hydroxide.

Microaerobic

＊ 0.01 M Ferric Quinate:

FeCl3 .....................................0.27 g

Quinic Acid (Sigma Q-0500)................0.19 g

Distilled water...........................100.0 ml

Dissolve and autoclave at 121C for 15 minutes.

＊ Wolfe's Vitamin Solution:

Available from ATCC as a sterile ready-to-use liquid (Vitamin Supplement, catalog no. MD-VS).

Biotin....................................2.0 mg

Folic acid................................2.0 mg

Pyridoxine hydrochloride..................10.0 mg

Thiamine . HCl............................5.0 mg

Riboflavin................................5.0 mg

Nicotinic acid............................5.0 mg

Calcium D-(+)-pantothenate................5.0 mg

Vitamin B12...............................0.1 mg

p-Aminobenzoic acid.......................5.0 mg

Thioctic acid.............................5.0 mg 2

Distilled water...........................1.0 L 3

* Wolfe's Mineral Solution:

Available from ATCC as a sterile ready-to-use liquid (Trace Mineral

Supplement, catalog no. MD-TMS.)

Nitrilotriacetic acid.....................1.5 g

MgSO4 . 7H2O ..............................3.0 g

MnSO4 . H2O ...............................0.5 g

NaCl......................................1.0 g

FeSO4 . 7H2O ..............................0.1 g

CoCl2 . 6H2O ..............................0.1 g

CaCl2 .....................................0.1 g

ZnSO4 . 7H2O ..............................0.1 g

CuSO4 . 5H2O ..............................0.01 g

AlK(SO4)2 . 12H2O..........................0.01 g

H3BO3 .....................................0.01 g

Na2MoO4 . 2H2O.............................0.01 g

Distilled water...........................1.0 L

Add nitrilotriacetic acid to approximately 500 ml of water and adjust to pH 6.5 with KOH to dissolve the compound. Bring volume to 1.0 L with remaining water and add remaining compounds one at a time.

基因改造承載質體pUMG與相關方法

改換成pRK415做為改造原料(成品：pKML)

加上Apr之類的抗性篩選因子

若AMB-1已寄送到，則可直接開始測定日本原先Mms13做為anchor之實驗的construct可正常表現與否

加上Luc或EGFP之類的基因，或加上適當的anchoring protein，測定大腸桿菌內表現活性。

加上Pmsp3 (超強promotor)

不能的話則先用Biobrick材料來test

magnetospirillum magneticum (AMB-1)菌種培養試驗

AMB-1（日本教授2001 paper裡面用的。Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1）

Medium Receipt(protocol):

1 liter of magnetic spirillum growth medium (MSGM) contains….

 Distilled water 1.0 L

 10 ml of Wolfe’s vitamin solution

 5 ml of Wolfe’s mineral solution

 2 ml of ferric quinate solution (0.27 g of FeCl3 and 0.19 g of quinic acid in 100 ml water)

 0.68 g of KH2PO4

 0.12 g of sodium nitrate NaNO3

 0.74g of succinic acid

 0.1 g of sodium thioglycolate

＊ Enriched MSGM contains

 0.02% polypeptone

 0.01% yeast extract

 0.005% L-cysteine instead of sodium thioglycolate.



All of the media used were supplemented with 5 mg/ml tetracycline

pH 6.75 with aqueous sodium hydroxide.

Microaerobic

＊ 0.01 M Ferric Quinate:

FeCl3 .....................................0.27 g

Quinic Acid (Sigma Q-0500)................0.19 g

Distilled water...........................100.0 ml

Dissolve and autoclave at 121C for 15 minutes.

＊ Wolfe's Vitamin Solution:

Available from ATCC as a sterile ready-to-use liquid (Vitamin Supplement, catalog no. MD-VS).

Biotin....................................2.0 mg

Folic acid................................2.0 mg

Pyridoxine hydrochloride..................10.0 mg

Thiamine . HCl............................5.0 mg

Riboflavin................................5.0 mg

Nicotinic acid............................5.0 mg

Calcium D-(+)-pantothenate................5.0 mg

Vitamin B12...............................0.1 mg

p-Aminobenzoic acid.......................5.0 mg

Thioctic acid.............................5.0 mg 2

Distilled water...........................1.0 L 3

* Wolfe's Mineral Solution:

Available from ATCC as a sterile ready-to-use liquid (Trace Mineral

Supplement, catalog no. MD-TMS.)

Nitrilotriacetic acid.....................1.5 g

MgSO4 . 7H2O ..............................3.0 g

MnSO4 . H2O ...............................0.5 g

NaCl......................................1.0 g

FeSO4 . 7H2O ..............................0.1 g

CoCl2 . 6H2O ..............................0.1 g

CaCl2 .....................................0.1 g

ZnSO4 . 7H2O ..............................0.1 g

CuSO4 . 5H2O ..............................0.01 g

AlK(SO4)2 . 12H2O..........................0.01 g

H3BO3 .....................................0.01 g

Na2MoO4 . 2H2O.............................0.01 g

Distilled water...........................1.0 L

AMB-1的替代品：magnetospirillum magnetotacticum (MS-1) magnetospirillum

Still aiting the author of the 中正大學's paper entitled"2009磁鐵細菌（地球磁場的領航員" to share his protocol in this paper with us, who is the 畢業研究生潘信凱。

And we had asked the director professor of this paper in the 中正大學, he promised to sent the strain MS-1 in two days(5/31-6/2) So right after we receive the bac MS-1 and the protocol from 學長, we'll start to culture MS-1 immediately.

寫信事宜

以下是目前最新進度0531，隨時更新！

要菌的進度（因為採買上的困難，先以跟別的實驗室要為主）：

我們目前最想要的菌是AMB-1（日本教授2001 paper裡面用的。Effects of growth medium composition, iron sources and atmospheric oxygen concentrations on production of luciferase-bacterial magnetic particle complex by a recombinant Magnetospirillum magneticum AMB-1），但於兩週前就已寫信兩三次給日本教授跟他們要AMB-1，但都沒回信仍石沈大海中QQ

所以我們決定跟中正大學的陳建易教授要菌（2009磁鐵細菌（地球磁場的領航員）但他們用的是類似的strain──MS-1。但因為這篇paper還在保護期不公開內容，很久前我便跟這篇碩士論文的學長聯絡，學長表示這兩天會寄給我paper。而五天前陳教授也很熱心的打電話給我，說可以給我們MS-1，也會幫我們寫信給德國大學要AMB-1！陳教授請他的助理寄了，這兩天應該會收到MS-1。

配置Medium（MSGM）的進度：

我們預定使用AMB-1，所以我們的protocol參考日本教授2001那篇為主，但是iGEM實驗室現在還缺滿多需要的藥品，學長建議我們直接去買現成的medium，並推薦我們以下兩種：

ATCC medium1653 http://www.atcc.org/Attachments/2856.pdf

BCRC medium 610 https://www.bcrc.firdi.org.tw/BSAS_cart/controller?event=SEARCH&bcrc_no=17316&type_id=3&keyword=Magnetospirillum

內容物看起來日本教授的protocol和ATCC（國外廠商）賣的比較像，但運送時間會比較久起碼兩週以上。而BCRC（台灣）賣的和目前我們的protocol相較則是缺滿多東西，但缺的東西比較好找，可能就我們買了自己再加進去。

其實也不用堅持一定要和日本那些不回信的教授（= = +）的protocol一樣，因為那篇是改良的protocol，照著做只是可以提昇菌或BMP的產率而已。since我們的目標不是養出超級多的磁菌和BMP，我們只要菌養的出來、菌又生的出BMP、BMP又有活性這樣就夠了。而市面上現成的medium和protocol差別只在於一些營養成分和iron source的不同，學長也說菌種類似，medium只要差不多其實都養的出來。我們也可以在養的過程中做修正與改良。

但有鑑於目前AB組都急迫的要開始養菌，所以我們先拿這兩天會到的中正MS-1來養，protocol也先參考中正大學那篇，或者購買BCRC的medium再做修正。所以最快一個禮拜內就可以開始養菌了，而菌在日本教授那篇裡面最理想的狀況需要4天長成（有關鍵的L-cystein情況下, which we and the 現成medium don’t have@@）。所以最快最快十天，六月中左右AB組就可以開始進行實驗了。

給C’的組員：

如果上面有任何不懂的……快回去做功課！！ =”= 讀paper！至少讀我等等寄給大家那篇日本大學 AMB-1的protocol裡我劃的重點，非常簡單！看過就會對medium的protocol有概念了。

C組(Panel C) [趨磁菌基因浮水印計畫]

基因工程編碼解碼設計

Adleman實驗的終極應用：雙重防護密碼+編碼系統

2010年香港中文大學"Bioencryption by recombination"

重要領域：DNA computing

電子書：6th International Workshop on DNA Based Computers

電子書：7th International Workshop on DNA Based Computers

電子書：8th International Workshop on DNA Based Computers

電子書：9th International Workshop on DNA Based Computers

電子書：14th International Meeting on DNA Computing

電子書：13th International Meeting on DNA Computing

電子書：DNA Computing and Molecular Programming--15th International Conference

電子書：Theoretical and Experimental DNA Computation

電子書：DNA computing models

密碼學(cryptography)

各類編碼方式研討

最適DNA之編碼方式

台大陳君明老師

密碼學課程講義

抽象代數導論

Main topic

B組(Panel B) [神經免疫反應調控]

培養microglia、macrophage

in vitro testing(關鍵核心實驗)

cytokines releasing amount measurement

intracellularity fitenss measurement

Listeria Monocytogene genes

要plasmid《快！》

利用homologous recombinant進入bacteria genomic DNA中

跨段落PCR：檢驗insertion的邊界是否正確

截切方式檢驗insertion的成功與否

篩選(耗時)

clone基因進入pKML並表現於AMB-1上

選promotor！

中研院

國外?《快上加快，不然會來不及！！！》

Hpt (G-6-P importase)

LLO (lyses phagosome membrane)

InlA (induces phagocytosis)

胞內共生機制與資料查詢

電子書閱讀

Phagocytosis of Bacteria and Bacterial Pathogenicity

Bacterial Evasion of Host Immune Responses

Bacterial Invasion of Host Cells

Central Nervous System Diseases and Inflammation

A組(Panel A) [磁控發光蛋白機制]

蛋白質工程設計

發光閃爍性質進階測試(核心實驗步驟)

split-lucierase complementation

FRET (using YFG & GFP as flanking sequence of Mms13)→detect proximity of N- and C-

蛋白質工程進行(modeling結束後；或是結構測定相關實驗結束後)

cloning操控：加長helix(?)

Directed Mutagenesis:Oligonucleotide - directed method

genetically structure template

Protein model

model2

model1

蛋白質結構測定(在modeling結果不滿意時採行之)

剔除某些片段以進行蛋白質功能結構測試(證實modeling結果)

"helix" knock-out

"turn" modification

Anticipated Results

Comfirmation & Observation Paradigm

向日本詢問：當年Mms13結構判定實驗，與概念確定的過程

Mms13蛋白質結購模擬

"Membrane Protein" Coarse-Grain Modeling

Use possible modeling program for Mms13

Find structures similar to Mms13 in data bank

CHARMM

ExPASy tertiary structure modeling tools

PoPMuSiC

SOSUI(transmembrane helix prediction)

Phyre

Phyre 2

Intensive

Normal

Phyre(original)

Mms13--145 a.a. version

Mms13--124 a.a. version

SWISS-MODEL

Luciferase

Mms13 no template