行政院國家科學委員會專題研究計畫 成果報告
融合分子生物科學與工程之生物分子工程學程研究(2/2)
計畫類別： 整合型計畫
計畫編號： NSC94-2522-S-011-001-
執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日 執行單位： 國立臺灣科技大學化學工程系
計畫主持人： 李振綱
共同主持人： 楊銘乾，林陳涌 計畫參與人員： 簡良榮 楊佩芬
報告類型： 完整報告
處理方式： 本計畫可公開查詢
中 華 民 國 95 年 10 月 30 日
一、 中文摘要(生物分子工程；工程與生物；生物技術；生物分子工程實驗；
技職教育)
整合台灣科技大學化學工程及高分子工程學系大學部及研究所原有之生物相關課 程，設計規劃一「生物分子工程學程」，將學程課程內容設計為建立在本校技職教育體 系同學所具之生物基礎上，結和化學相關工程與生命科學之跨領域教育，幫助工程背 景學生學習生命科學相關基礎學科，並教導技職教育體系學生利用已具備之化學相關 工程背景知識，以實作課程學習解決生物領域相關工程問題，使學生具備有解決生物 科技產業化可能面臨之問題的能力，以因應未來生物科技相關產業快速發展的世界。
本計畫執行第一年已有二十名大學部學生，第二年有十名大學部學生、十五名研究生，
分別於寒暑假期間參加基礎生物分子工程實驗、進階生物分子工程實驗及生物分子工 程實務專題研究。規劃撰寫完成基礎及進階生物分子工程實驗兩本教學手冊，完成生 物分子工程教學網站。
二、英文摘要
Most of the undergraduate students of National Taiwan University of Science and Technology have no enough biological science knowledge because there is no biology related courses offered during their study in the senior high technical-school. In order to extend student’s future career in the field of biotechnology-related industry, in this project we proposed a bio molecular engineering program for our students mostly from Department of Chemical Engineering and Department of Polymer Science because those students have better chemistry background. Students in this program are required to take Biology, Biochemistry, and Biotechnology courses in order to intensify their lacked modern biotechnology knowledge. In addition, students are required to learn basic hand-on bio-laboratory techniques by taking Fundamental Biomolecular Engineering Laboratory. After the students acquired enough modern biotechnology knowledge and experimental techniques, the students are required to accomplish a biological process project by using integrated engineering and biology knowledge. In this proposed biomolecular engineering program project, some basic lab works and bioprocess projected are designed and the related experimental procedures and background knowledge will be written to provide students a better learning process. We hope this program will prepare engineering students for entering the booming modern biotechnology industry.
Keywords: biomolecualr engineering; integrated engineering and biology education, technical
目錄
一、報告內容 ---
3
前言 --- 3
目的 --- 3
方法 --- 3
二、成果 --- 5
參考文獻 --- 6
成果自評 --- 7
附錄 --- 7
二、 報告內容 前言
近年來隨著人類基因解碼的完成，國內外醫藥生物相關技術產業都蓬勃發展，因 應此趨勢的發展，國內各大學紛紛成立生命科學或生物技術相關系所，培育生物科學 研究人才，少有學校培育能與生物科技結合的跨領域人才，若無互補性跨領域之工程 背景人才參與，生物科技產業之製造程序無法有效控制管理，生產製造無法最適化，
技術產品不易推廣應用，產品商業化的目的無法順利達成，因此培育能與生物科技互 補結合的跨領域工程人才參與生物科技產業的發展是十分迫切。
目的
一般技職教育體系中之科技大學工學院的學生因為在高職或工專時期已經分科，
在高工或工專中少有生物課程，大多數學生的生物科學的知識多只停留在國中程度，
因此本研究計畫之目的，在規劃可以兼顧科技大學工程學院學生的生物背景知識與未 來生涯發展需求的生物科技課程，使工學院大學部學生能夠對生命科學及生物技術的 基本知識和未來發展有基本的認識與了解，使研究所的研究生能夠具有發覺並解決與 生物相關的工程問題的能力。
方法
生物科技的領域十分廣泛，從農業、食品、一直到醫療都包括在其中，但回歸到 生物的基本結構來看，其實都是在探討研究相同的生物分子如 DNA、 RNA、蛋白質、
多醣類等，因此本教育計畫是以生物分子為主要基礎來規劃、設計工程生物的整合教 育課程，本計畫之總體目標為建立融合分子生物科學與工程之生物分子工程學程教 育，生物分子工程(biomolecular engineering) (有關生物分子工程的重要可考 ”Recent progress in biomolecular engineering” Biotechnol. Prog. 2000, 16, 2-16.)是融合了分子生 物技術、細胞科學、及工程的一門新且重要的領域，此領域主要是探討如何利用生物 分子（如核酸、蛋白質等）及生物分子程序（生物分子之間交互作用、催化反應等）
去發現高價值的生物分子，或者是如何去利用各種細胞做為高價值的生物分子的生產 工廠，更有效率的產生出此高價值的生物分子，所運用的生物知識與技術主要是包括 生物化學、分子生物學、蛋白質化學，基因重組技術，蛋白質工程等，所運用的工程 知識與技術則包括了動力學、動量、質量、及能量的輸送現象。由於生物分子工程與 化學工程領域息息相關，國外不少化學工程系所(U. Illinois, Urbana Champagne; Cornell U.)已改名附加上生物分子工程，朝向生物分子工程方向積極發展。本校之化工及高分 子工程，其必修科程中已經包括有生物分子工程中所需之工程與技術知識，因此針對 本校工程學院之化工、高分子等領域的學生規劃設計生物分子工程學程，以期增加學 生在生物科技上之知識與技能，拓展其就業領域，並為國家生物產業之發展提供所需
生物分子工程學程課程規劃如下:
科目總表:
A. 大學部需修滿學程課程 13 學分，包括專業科目生物工程 (3 學分)及生物分子 工程基礎實驗(1 學分)兩門，基礎課程包括師大生科系支援教授之普通生物 學、生物化學，基礎分子生物學、生物技術(各三學分)至少三門。
B. 研究所學程則需修滿學程課程 10 學分，包括專業科目生物分子工程 (3 學分) 及生物分子工程實驗(1 學分)兩門，及其他專業相關課程應用微生物、基因重 組技術、酵素工程至少兩門。
大學部及研究所學程課程安排如表一
表一:學程課程安排時間表
學期 課程 一下 普通生物 二上 生物化學
二下 生物化學 生物技術
三上 基礎分子生物學 三下 生物分子工程基礎實驗 四上 生化工程 研究所上 生物分子工程、酵素工程 研究所下 應用微生物、基因重組技術 研究所暑假 生物分子工程實驗
在此課程中除學科外，亦在寒暑假期間利用原有之實驗室安排有大學部之生物分子工
程基礎實驗及研究所之生物分子工程實驗，建立學生基礎之生物工程實驗技術，此外 技職教育首重實作，因此皆安排有必修之實務專題，在此大學部學程中參與學生需以生物工程相關專題，在兩學期中完成本校必修之專題研究課程，此學程之評量重點之 一在於學生之專題研究成果：
實驗專題成果展示
本學程計畫中最主要的部分是增添本校所最欠缺的生物相關實驗規劃，此實 驗內容包括兩部分一為基礎核心的生物分子實驗，另一為需做出實物成品的 應用專題實驗，因此在學期末課程結束時將要求學生以書面專題實驗報告 外，也要以口頭報告、與全體參與學生討論的方式呈現其學習成果，並以此 做為此學程的評量特色之一。
此外也將編撰學程教材，其成果也將上網展示
有關『生物分子工程學程』的課程，將由任課教師負責教材編撰，亦將推動 教材上網，透過此方式普遍傳達生物工程慨念。並將架設台科大生物分子工 程學程網站，增進國內外此學程之交流。
二、 成果
第一年計有本校四年制及二年制化工系學生二十名加入學程，93 年度寒假期間 (1/20-1/26/2005)全天進行生物分子工程基礎實驗，實驗內容及時程安排如下表
1/20 (四) 1/21 (五) 1/24 (一) 1/25 (二) 1/26 (三) A 組
1. Pipet 使用4 2. 分光光度計
使用5 3. 滅菌及無菌
流程操作2 4. 培養皿製作
1、劃菌及勾 菌3
註: 滅三角搖瓶 勾含 EGFP 之 菌株
A 組 1. diauxic growth
生長曲線1 2. 菌液離心固液
分離3
3. 離心機使用3 註:勾含
pET30b(+)之菌株
A 組 1. 超音波打
破菌體1 2. 蛋白質電
泳製作2 3. 質體 DNA
萃取3 4. DNA 電泳4
註:純化 buffer 之配置
A 組 1. 離子交換層
析分離1 2. 金屬層析分
離2
3. 蛋白質電泳 製作3
註:勾含
DAOase 之菌株 滅三角搖瓶
A 組 1. 放大培養
DAOase 菌株
1
2. 酵素活性測 定2
註: 勾含 EGFP 之 菌株
討論時間 討論時間 討論時間 討論時間 討論時間
B 組
1. Pipet 使用4 2. 分光光度計
使用5 3. 滅菌及無菌
流程操作2 4. 培養皿製作
1、劃菌及勾 菌3
註: 滅三角搖瓶 勾含 EGF 之 菌株
B 組
1. 搖瓶之菌體生 長曲線1 2. 菌液離心固液
分離3
3. 離心機使用3 註:勾含
pET30b(+)之菌株
B 組
1. 超音波打 破菌體1 2. 蛋白質電
泳製作2 3. 質體 DNA
萃取3 4. DNA 電泳4
註:純化 buffer 之配置
B 組
1. 離子交換層 析分離1 2. 金屬層析分
離2
3. 蛋白質電泳 製作3
註:勾含
DAOase 之菌株 滅三角搖瓶
A 組 1. 放大培養 DAOase 菌株 2. 酵素活性測 定2
註: 勾含 DAOase 之菌株(B 組)
第二年計有本校四年制及二年制化工系學生十名加入學程，94 年度寒假期間進行生物 分子工程基礎實驗。
第一年學生完成實務專題研究計有:
1.環境因素對細菌纖維素之培養生產的影響
2.細菌纖維素之改質及其應用
其中以細菌纖維素分離純化乳酸鍊球菌素之專題更獲得本系最佳專題評比之佳作獎。第一 年參加本學程之學生畢業後有兩位學生升入研究所，仍然以選擇生物工程相關領域為其論 文研究方向。第二年除十名大學部學生加入學程，更有十五名研究生參加寒假所舉辦之生 物分子工程實驗課程。其實驗內容包含:
Polymerase Chain Reaction (PCR)；Confirmation of PCR Amplification and Initialization of Expression Plasmid Construction；Restriction Enzyme Digestion；Expression Plasmid Construction; Transformation of Escherichia coli by plasmid DNA; Analysis of Bacterial Growth and Selection of Clones; DNA Isolation; SDS-PAGEImmuno/Western Blotting等九項實驗，因應本校國際化之推展並配合研究生中之外籍國際學 生，此課程皆以英語教學，及編撰英文實驗教材。
第二年學生進行中之實務專題研究計有:
1.重組枯草桿菌發酵生產透明質酸 2.醋酸菌生產細菌纖維素
3.重組枯草桿菌表現生產轉氨基醯氨基脢 4.光照對重組大腸菌生產 ALA 之影響 5.毛細管電泳定量分析醣成份
預計九十五年十二月可完成。
兩年內已完成大學部之課程規劃且已在執行中，實驗課程也已完成，生物分子工程基 礎實驗、生物分子工程實驗(英文版)實驗教材已編撰完成(如附件一)可推廣使用。實驗流程 安 排 及 學 生 分 組 ， 學 生 完 成 之 報 告 已 公 告 陳 列 在 所 建 構 之 生 物 分 子 學 程 網 頁 http://web.ntust.edu.tw/~ntustbiolab/。此外學程之專題研究題目介紹及學生專題研究題目之 分組相關資料也都呈現於網頁中。
三、參考文獻
1. Dewey D. Y. Ryu* and Doo-Hyun Nam; Recent progress in biomolecular engineering”
Biotechnol. Prog. 2000, 16, 2-16.
2. C. Komives, S. Rech, M. Mcneil, Laboratory Experiment on GENE SUBCLONING For Chemical Engineering Students. Chemical Engineering Education, Summer 2004, 212-221
3. V. Hatzimanikatis, D. Collins, S. Lawrence, S. Browning, K.H. Lee, Bioinformatics.Genomics, and the chemical engineering a perspective, Chemical Engineering Education Fall 2000, 346-349.
4. Kathryn A. Hollar, Stephanie H. Farrell, Gregory B. Hecht, Patricia Mosto, Integrating Biology and ChE at the Lower Levels, Chemical Engineering Education, Spring 2004
5. Ken K. Robinson, Joshua S. Dranoff, Christopher Tomas, and Seshu Tummala, Mass Transfer and Cell Growth Kinetics in a Bioreactor, Chemical Engineering Education, Summer 2002
6. Xuemei Li, Xiao Dong Chen, Matthew T. Hardin,Investigation into the Propagation of
Baker's Yeast: A Laboratory Experiment in Biochemical Engineering, Chemical Engineering
Education Summer 2004,
7. 黎耀基 我國生物科技產業與生技人材培育 科學發展月刊 29卷5期 307-313
四、成果自評：
計畫編撰之生物分子工程基礎實驗、生物分子工程實驗兩教材已完成，且經學生實習使用
過，可幫助學生對生物與工程領域之結合有深刻之瞭解。但所編之實驗若需繼續執行則需 校方提供材料費及助教協助方可供學程之長期維持與發展。計畫開授之課程也已開授，但 受限於實驗室空間及後續實驗材料費之不足，參與此學程計畫之學生人數無法成長。
五、附錄：
1.生物分子工程基礎實驗教材
2.生物分子工程實驗(英文版)實驗教材
台灣科技大學生物分子工程學程
基礎生物分子工程實驗教材
Biomolecular Engineering --- A Fundamental Laboratory Course
National Taiwan University of Science and Technology Department of Chemical Engineering
Biomolecular Engineering Program
2005 October
目 錄
1. Pipet 使用 ...1
2. 分光光度計原理及使用...3
3. 培養皿製作、劃菌及勾菌...4
4. 菌株生長曲線之測定...5
5. 革蘭氏染色法...6
6. 質體 DNA 萃取及洋菜膠體電泳...7
7. 蛋白質電泳...8
8. 蛋白質離子交換層析分離...10
9. 蛋白質金屬層析分離...11
10. 酵素活性測定分析(initial rate of absorbance increase) ...12
1.Pipet 使用
微量吸管 (micropipet) 是進行生化實驗或分子生物學實驗的必備工具，然而使用方法的正確與 否，以及微量吸管的準確性，都直接影響了實驗結果的正確性，故請對你持有之微量吸管作深入的 認識。
A. 基本使用方法
1.選擇適當的 Pipetman：不同型號的微量吸管，各有其吸取體積範圍，請依取用溶液體積取用適 當的微量吸管。
2.設定體積：設定体積時，由低旋轉至高值，須先超越所欲設定值至少三分之一轉後，再反轉 至設定值；由高旋轉至低值，則直接轉至設定值即可。請勿將体積調整圈轉到超過 最低或最高的使用範圍。
3 套上微量吸管頭，吸取溶液： 吸取溶液時，尖端請先套上微量吸管頭 (tip)，P1000 使用藍色微量 吸管頭，P100 使用黃色微量吸管頭，P10 使用白色微量吸管頭。將 按鈕壓至第一段，儘可能保持微量吸管垂直，將微量吸管頭尖端浸 入溶液，再緩慢釋放按鈕。釋放按鈕不可太快，以免溶液衝入吸管 柱內而腐蝕活塞。
4. 釋放溶液：將微量吸管頭與容器壁接觸，慢慢壓下按鈕至第一段，停一兩秒再壓至第二段 把溶液完全壓出。
A, 保持微量吸管垂直，將按鈕壓至第一段；B, 微量吸管頭尖端浸入溶液，緩慢釋放按鈕；C, 持微量吸管垂直，將微量吸管頭與容器壁接觸，慢慢壓下按鈕至第一段；D, 壓至第二段把溶液 完全釋放出；E, 釋放按鈕回原狀。
B.使用注意事項
1.勿將微量吸管本體浸入溶液中。
2.吸取黏度高之試液，請先將微量吸管頭尖端以刀片或剪刀將出口切大，並先行預潤後再吸取。
3.微量吸管的任何部份切勿用火燒烤，亦不可吸取溫度高於 70℃ 的溶液，避免蒸氣侵入腐蝕活 塞。
4.將調整後體積的 Pipetman 使用過後，應調整回該 Pipetman 的最大容許值，避免 Pipetman 內彈 簧彈性疲乏。
C.實驗方法步驟
1.請取六支微量離心管，分別標上 A~F。
2.以正確使用方法，依下表所列體積吸取不同溶液至各管中。
單位 µL A B C D E F
Solution 1 Solution 2 總體積
3.將管蓋蓋好，置入微量離心機，離心數秒使溶液混合並集中於管底。
4.將適當微量吸管之體積調整圈調至總體積處，以乾淨的微量吸管頭吸取管中的溶液，並檢查：
(i) 微量吸管頭尖端是否剛好充滿溶液或留有一些空間？
(ii) 離心管中是否有溶液殘留？
2.分光光度計使用
A.吸光度測定流程
1.打開右側方之電源開關。
2.點選桌面上之 Spectra manager 開啟程式。
3.進入畫面之 Environment，點選 Hardware setting 將 Deuterium 取消(如果使用波長大於 340nm)。
4.隨之點選畫面之 Fixed wavelength measurement 進入定波長測定。
5.在點選 measurement 之 parameters，將 wavelength 該為欲測之波長，之後按 OK 即可。
6.將空白樣品放入待測槽中，按下 Autozero 歸零即可偵測待測物之吸收值大小。
B.使用注意事項
1.測定 UV 範圍(<340nm )請用石英測光管。
2.測定之吸光值若超過 0.8，請將溶液稀釋後再測。
3.光度計上請勿放置任何物品，尤其是溶液。亦請勿在光度計上操作實驗。
4.放入測光管前，請將測光管外壁擦拭乾淨，不可有任何液體殘留。
5.使用後測光管必須立即以蒸餾水沖洗乾淨，拭乾後置回原位。
6.使用完畢務必關掉燈泡及電源開關。
C.測定溶液之吸光值
1.秤取 1mg 之牛血清蛋白 BSA 溶於 10mL 蒸餾水，當作 BSA 標準液。
2.將 BSA 標準液以蒸餾水稀釋成不同濃度(0.05、0.01、0.005、0.001、0.0005、0.0001 mg/mL) 3.再將分光光度計定波長設為 OD280。
4.將上依步驟混合之液體靜置，即放入分光光度計進行偵測不同蛋白質濃度之吸光值。
5.紀錄不同濃度所得之吸光值，並以濃度與吸光值作圖，建立 BSA 檢量線，觀測其線性迴歸 R2~0.999，即可得知操作者 pipet 使用是否正確及分光光度計之使用。
3.培養皿製作、劃菌及勾菌
A.培養基之配製
LB agar plate : 1% (w/v) Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl，以 5 N NaOH 調至 pH 7.0 後，加入 1.5% (w/v) Bacto agar
1.培養基配置如上所示，配製完後以濕式滅菌法滅菌約 30 分鐘。
2.待冷卻至 50℃左右於無菌操作台內加入所需抗生素，同時攪拌均勻，並依次倒入培養皿 中即可。
B.劃菌
1.取一個加有 Kanamycin 之 LB plate (LB+Kan)，在培養皿底部標明組別及日期，並寫上菌株名稱。
2.將生長有菌株之培養皿蓋子拿起，以滅菌牙籤輕輕碰觸刮下菌落，並將培養皿蓋子置回。
3.打開空白的 LB+A plate，將沾有菌落的滅菌牙籤於其上輕輕連畫數條橫線 (Streak 1) 後，將培 養皿蓋子置回。
4.將培養皿旋轉約 60 度，畫第二次線 ( Streak 2)。
5.重複上一步驟之操作，畫第三次線 (Streak 3)。
6.將培養皿倒置，於 37℃ 培養箱中培養過夜
C.勾菌培養
LB 培養液：1% (w/v) Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl，以 5 N NaOH 調至 pH 7.0 後，以溼熱法滅菌。
1.取一支 15 mL 試管，並以 pipet 吸取 3 mL LB 培養液於試管中，請寫上菌株、標明組別及 日期。
2.打開試管蓋 (一手握住試管，並以拿著竹籤那隻手的小指旋開蓋子)，將管口在酒精燈上過 火。將沾有菌落的竹籤伸入培養液中並輕敲管壁使菌落脫離。將管口再次在酒精燈上過火後，
將蓋子置回。
3.將試管置於 37℃ 培養箱或水浴中震盪培養過夜。
※與細菌接觸過的任何容器或培養基，使用後須經過滅菌才可丟棄。
Streak 1 Streak 2 Streak 3
4.菌株生長曲線之測定(diauxic growth)
When exposed to glucose + lactose, E. coli does not consume lactose until glucose is exhausted, resulting in two exponential growth phases separated by a lag. This is called the diauxie or “double growth.” Diauxie occurs because synthesis of lactose permease and β-galactosidase is somehow abolished in the presence of glucose.
1.取兩個 250 mL 之滅菌過三角錐瓶，利用吸量管吸取 30ml 滅菌後之 LB 培養基於此兩三角錐 瓶，其中一個加入滅菌過之 glucose 溶液(0.5%)，另一個加入等濃度之 glucose(0.5%)和 lactose(1%)，請標明組別。
2.取昨天接種並經過夜培養的菌液 1 mL，加入三角錐瓶中，混合均勻後，吸出 1 mL 於微量離 心管中，置於冰浴中。 三角錐瓶則置於 37℃ 震盪培養。
3.其後每隔約 60 min 吸出 1 mL 菌液，直至菌株生長持平，吸出之菌液皆暫置於冰浴中。
4.所有取樣之樣品，皆利用分光光度計測不同時間吸出菌液之 OD600，請以 LB 培養液作為 Blank。
5.最後將菌液以高速離心方式進行分離。
6.以時間為 X-軸，OD600作圖，比較此兩瓶生長曲線之不同。
5. 革蘭氏染色法
A.原理
鑑別染色需用經熱固定之抹片，且連續使用三種化學藥劑。最初試劑是所謂初染劑，其目的使細菌 著色。為形成顏色對比，第二試劑為脫色劑。依細胞化學組成之不同，脫色劑可完全除去初染或僅 除去部份細胞構造之顏色。最後試劑是謂複染劑，與初染呈對比顏色。脫色時若初染未洗去，則複 染不能吸收，細胞與其組成將保留初染顏色。若初染脫色，則細胞組成即吸收複染之對比顏色。因 此細胞之形成或其構造，可依保留之染色劑而鑑別之。用於細菌學上最重要之鑑別染色法是謂革蘭 氏染色。此法乃微生物分類與鑑別之主要工具，可將細菌區分為兩大組，即革蘭氏陽性菌與革蘭氏 陰性菌。本法使用四種不同試劑，茲將其作用機轉分別簡述如下：
初染劑：初染劑乃結晶紫(crystal violet)，係步驟中最先使用之試劑，使細菌染成紫藍色。
媒染劑：乃革蘭氏碘液(Iodine)，可與初染劑結合而形成不溶性複合物。所形成之結晶紫碘（CV-I）
複合體藉以加強染料顏色，此時所有細菌均成紫黑色。
脫色劑：乃 95%乙醇，此試劑具有脂溶性與蛋白質脫水劑之雙重功能。其作用依微生物細胞壁 所含脂肪濃度而定。格蘭氏陽性菌之脂質含量低乃保留 CV-I 複合體之主要因素因少量 之脂質經乙醇作用，立刻溶解，形成細胞壁之細孔，但此細孔隨即因乙醇之脫水作用而 封閉，以致緊密結合之初染劑不易移去，終於保留紫色。革蘭氏陰性菌細胞壁中之外層 含高量之脂質，易為乙醇溶解，形成大孔。即使引起細胞壁蛋白之脫水也不易封閉該孔，
因此有利於 CV-I 複合體之釋出，致使菌體成無色。
複染劑：最後試劑為番紅(safranin o)，凡經上述脫色之細菌，被此染料染成紅色。因僅革蘭氏陰 性菌發生脫色，故於此步驟能吸收複染劑。革蘭氏陽性菌則保留初染時之紫色。
B.實驗步驟 1.拭淨玻片。
2.以無菌操作，分別將 Escherichia coli 及 Streptococcus thermophilus 及混合菌株置於抹片上，於 空氣中乾燥，熱固定後備用。
3.將塗抹覆蓋結晶紫，並靜置一分鐘。
4.以蒸餾水輕輕的洗。
5.碘液沖掉多餘的水分然後以碘液覆蓋載玻片放置 60 秒。
6.自來水沖洗。
7.酒精進行脫色至脫色液無色。
8.自來水洗掉多餘的酒精。
9.再以番紅複染 45 秒。
10.以吸水紙吸乾，並用油鏡觀察。
C.注意事項
※染色時，脫色步驟可影響抹片之製備成敗與否。切記脫色過度時可使初染流失，使革蘭氏陽性 菌成革蘭氏陰性菌。但脫色不足，則不能完全除去 CV-I 複合物，結果是革蘭氏陰性菌成革蘭 氏陽性菌；
※將玻片以蒸餾水充分輕洗，以除去剩餘之染液；
※宜使用未超過 24 小時之新鮮培養，置備抹片。因菌齡老化，尤以革蘭氏陽性菌易失去保留初 染之能力，而顯示出錯誤的結果。且部份菌體染呈紫色，部份呈紅色。
6.質體 DNA 萃取及洋菜膠體電泳
A.質體 DNA 萃取試劑藥品(鹼溶裂法)
細胞懸浮溶液 TE buffer：25mM Tris-HCl, 10mM EDTA, pH8.0 細胞分解溶液 Lysis buffer：0.2N NaOH, 1% SDS
中和緩衝溶液 Neutralization buffer： 5M potassium acetate
B.鹼溶裂法方法步驟
1.將含有質體 pET30b(+)之菌株勾取置 3mL 的特定培養基(LB+Kan)中，於 37℃、170rpm 震盪培 養約 16 小時。
2.高速離心收取細胞(6000rpm ,1min) 3.加入 200µL TE buffer 將細胞完全懸浮。
4.加入 200µL Lysis buffer，然後輕輕翻轉 10 次。
5.加入 250µL Neutralization buffer，然後輕輕翻轉 10 次。
6.高速離心(12000rpm,5~10min)，取上清液於新的微量離心管。
7.加入 500µL 異丙醇 IPA 將質体 DNA 沉澱，高速離心(12000rpm,5~10min)，移除上清液。
8.加入 500µL 70% 酒精輕洗質体 DNA 沉澱，高速離心(12000rpm,5~10min)，移除上清液。
9.將質體 DNA 靜置烘乾，加入 20µL TE buffer，則可獲得純化後之質体 DNA。
C.電泳試劑藥品
洋菜膠 (agarose, low EEO) 1×TAE 電泳緩衝液
10× 追蹤染劑：0.25% bromophenol blue, 0.25% xylene cyanol, 0.1 M EDTA, 50% glycerol。
SYBR green stock solution (100X)
DNA 標準分子量 DNA/HindIII fragments：包含 8 個片段 23.1, 9.4, 6.5, 4.3, 2.3, 2.0, 0.56, 0.125 kb，濃度為 0.5 µg/µL，以下簡稱 λMr。
D.電泳方法步驟
1.製備一片 8-well 1.2% agarose gel：秤取適量 agarose 粉末約 0.3g，加入 1×TAE 25 mL，以 微波爐加熱溶解後，注入鑄膠器中。
2.取不同大小之 DNA 分別加入 2µl SYBR Green 及 2µl loading dye，靜置於暗處約 30 分鐘，在 依序注入 well 中進行電泳。
3.以 100V 進行電泳，待追蹤染劑 bromophenol blue 行進至膠體三分之二處時，關閉電 源，取出膠片，以 UV transilluminator box 觀察色帶位置，並記錄結果。
4.與標準分子量比較，估計各 DNA 片段之分子量。
7.蛋白質電泳
A.原理
電泳的高解析力使其成為生化技術中最有效力的一門分析利器，以下簡略說明各種電泳的形式及其 應用。
1.不連續膠體電泳 (disc-PAGE) 是以原態蛋白質進行電泳，一般用作純度檢定或活性分析，SDS- 膠體電泳(SDS-PAGE)則用為次體分子量之測定，梯度(gradient) 電泳則可輔助原態蛋白質分子 量之決定。 結合 SDS 及梯度兩種特性的 SDS-梯度膠體電泳，其解析力最高。
2.電泳形式 早期使用圓柱狀膠體，演變成直立式的平板膠片 (16 × 18 cm)，最後改成迷你電泳 膠片 (8 × 10 cm)，電泳時間只約一小時，而其解析力不變。現在柱狀膠體電泳只用在等電焦 集法，以進行二次元電泳；而大型平板電泳則多用在製備式電泳，一般電泳大多以迷你膠片進 行之。
3.膠體材質 的種類很多，但用在蛋白質者則以 聚丙烯醯胺 (polyacrylamide) 為主；聚丙烯醯胺 電泳是蛋白質應用最廣的電泳分析方式。也有少數使用洋菜膠體 (agarose gel)，多用在測定 pI 較廣的異構脢酵素群。
4. 泳動率：在 pH 8.8 的電泳條件下，大部分 pI 小於 8.8 的分子均能往正極泳動；蛋白質的 泳動率與所帶電荷成正比，而與其分子量成反比；若分子量一樣，但形狀越不規則，或體積越 鬆散者，泳動率越小。
B.儀器用具及藥品 1.迷你電泳槽 2,鑄膠器
3.鑄膠三明治組合：玻璃板 (8x10 cm)、白色氧化鋁板、Spacer 間隔條(0.75 mm) 、Comb 樣本齒 模(0.75 mm)
4.供電器 5.塑膠染色盒 6.旋轉搖盪器
7.試劑組：30% acrylanide mix 、1.5M Tris(pH8.0)、10% Ammonium persulfate、10% SDS 及 TEMED
泡靜置約 20 分鐘。
3.將製膠槽中之 Isopropanol 傾斜倒出，再依序混合表之成分注入製膠槽中，插入樣本齒模以形成 樣品槽。
4.將欲分析之樣品加入 3X loading dye 置於 95℃恆溫器中，加熱 10 分鐘；蛋白質標準液則只需 加熱 5 分鐘。
5.待加熱後之樣品冷卻後，再依序注入 SDS-PAGE 之樣品槽中，並以 110V 進行電泳分析，待藍 色染料色帶跑至膠片底部後即可關閉電源。
6.取出膠片組合，使玻璃板向上，先除去兩側間隔條，再以間隔條輕輕撬起玻璃，膠片則留在 白板上。切除焦集膠體，並在分離膠片的右上方截角做記號 (區別膠片的左右)。
7.將完成之電泳片置於 Staining buffer 中染色約 5 min，染色盤要加密蓋，置於旋轉平台慢速 50 rpm 搖盪之；要注意膠片是否完全沒入染色液中，否則會造成染色不均勻。
8.染色後小心把 CBR 染劑倒回原來瓶中，整塊膠片變成藍色，先用自來水洗掉殘餘的染色液，
再倒入約 20 mL Destaining buffer，加密蓋後再放回旋轉平台搖盪。染色脫色過程請戴手套，
否則會在膠片上留下指紋，而後再以照相系統存取結果即可。
8.離子交換層析分離
A.原理
各種蛋白質分子上可能帶有不同的電性，經過離子交換管柱中的帶電填充膠體，可依其分子帶電性 的差異而分離開來。
B.藥品試劑
1. Buffer A ：50 mM Na3PO4, pH 7.0
2. Buffer B ：50 mM Na3PO4, pH 7.0, 0.5 M NaCl 3. DEAE- cellulose
C.方法步驟
1.震盪 DEAE cellulose 膠體使之懸浮，小心倒入管柱中，讓膠體慢慢沈降，在沈降過程中隨時 加入 buffer A，勿使膠體乾掉。
2.待膠體沈降完全後，高度應在 0.5 cm 左右。膠柱以 buffer A 一直流洗，流速快慢可以不考 慮。
3.樣本的添加方法同上述膠體過濾法，當樣本全部沒入膠體後，收集流出液並以 buffer A 流洗 10 mL 後，去除膠體上方的緩衝液，但勿使膠體乾掉。
4.然後依序以 bufferB 溶離之，流洗約 1 mL。
5.收集衝提液，以進行 SDS-PAGE 電泳分析。
9.金屬層析分離
A.原理
若表現蛋白質上含有一段六個 His 的片段，而親和吸著劑膠體上接有鎳離子，此蛋白質會專一性地 結合到吸著膠體；洗去雜質後可用 imidazole 溶離純質蛋白質
B.藥品配製
1.金屬螯合親和層析膠體
2. Buffer A：50 mM Na3PO4, pH 8.0; 0.3 M NaCl; 20 mM imidazole 3. Buffer B：50 mM Na3PO4, pH 8.0; 0.3 M NaCl; 250 mM imidazole
C.方法步驟
1.親和層析膠體 0.5 mL 已經裝填在管柱內，成為一淡藍色膠柱。請把管柱架直在鐵架上，去除 下方的填塞物，用 buffer A 流洗 20 mL 後塞住出口，準備注入樣本。
2.除去膠面上方的緩衝液，並取出樣本慢慢用滴管加到親和膠體上，小心勿弄亂膠體表面；讓 樣本沒入膠體中。＊收集流出液
3.待樣本全部進入膠體後，關閉出口，再慢慢加入 buffer A，打開出口收集流出液；buffer A 共流洗 10 mL。
4.接著以 buffer B 流洗 1ml。
5.收集衝提液，以進行 SDS-PAGE 電泳分析。
10.酵素活性測定分析
1.取純化後之酵素及粗菌液至於冰水浴中待用。
2.分別依序加入
Reagent Stock Final Volume
H20 675µL
Potassium phosphate buffer, pH8.0 1M 0.1M 100µL D-alanine 0.3M 0.03M 100µL o-phenylenediamine/o-dianisidine 0.3% 0.03% 100µL
Horseraddish peroxidase 0.25U/µL 5U 20µL
Enzyme(DAOase) 5µL
3.將分光光度計開啟，並點選 Time course measurement，在選擇 Measurement 之 parameters 將參 數依下列更改 Photometric mode=abs；Response=quick；wavelength=453nm；End time=120sec；
Data Pitch=0.2sec；Display=auto。
3.利用分光光度計之動力學參數來偵測 OD453 之隨時間變化。
4.分別分析粗菌液、離子交換純化液及金屬層析純化液之間之活性差異。
A Practical
Laboratory Course of Biomolecular
Engineering
Molecular Cloning Techniques
Department of Chemical Engineering National Taiwan University of Science
and Technology
2006/2/13
Table of contents
1 : Polymerase Chain Reaction (PCR) ... 1 2 :Confirmation of PCR Amplification and Initialization of
Expression Plasmid Construction ... 3 3 : Restriction Enzyme Digestion... 5 4 : Expression Plasmid Construction ... 7 5 : Transformation of Escherichia coli by plasmid DNA ... 9 6 : Analysis of Bacterial Growth and Selection of Clones ... 11 7 : DNA Isolation ... 14 8 : SDS-PAGE ... 15 9 : Immuno/Western Blotting ... 18
Molecular Cloning Techniques
The main objective of this laboratory sessions is to introduce graduate students of Chemical Engineering to key techniques utilized in molecular biology and, specifically, molecular cloning. This group of sequential exercises is designed to take students through the natural progressions encountered when inserting a gene of interest in an expression vector for subsequent gene product expression. Students will learn key techniques such as polymerase chain reaction, expression plasmid construction, restriction enzyme digestion and gel electrophoresis as well as acquire useful tools such as bacterial transformation and DNA isolation. These essential applications form the foundation of molecular biology and will provide students with the research tools for examining gene expression in prokaryotic or eukaryotic systems.
Exercise 1: Polymerase Chain Reaction (PCR)
Polymerase Chain reaction (PCR) is a technique used by almost every biology laboratory in the world. This tool is used for the detection of gene products and gene amplification for both research and clinical diagnostic purposes. This technique is very simple and relatively inexpensive requiring two amplification oligonucleotides, a DNA polymerase, a mixture of stabilizing buffers as well as the DNA/RNA template for amplification and analysis. The premise behind PCR is simple, requiring three main phases or steps. In the first phase, the DNA template is denatured at a relatively high temperature (95°C).
The second step requires a lower temperature (50-65°C) for annealing of the oligonucleotides to subsequently amplify the gene/fragment of interest. The final phase is completed at the optimal temperature for polymerase activity (72°C) where the amplified product is elongated from the 3’ end of the primers. The time required for each step is dependent upon the length of the segment to be amplified as well as the source of genetic material (ex: plasmid DNA, genomic DNA, etc.). These steps are repeated in cycles (25-40 cycles/run) in order to amplify sufficient amounts of the product for analysis or applications such as subcloning. PCR has become an essential and reliable tool for basic research as well as clinical diagnostics.
Objective:
Using pEGFP-C1 as a template, students will use PCR to amplify the gene encoding for Enhanced green fluorescent protein (EGFP). The amplified fragment will be visualized using gel electrophoresis and subsequently used to construct a EGFP expression plasmid.
Materials:
pEGFP-C1 ---map on next page PCR Buffers (10X buffer, dNTPs) 5’and 3’EGFP oligonucleotides Taq polymerase
Sterile water PCR Tubes Pipettemen Pipette tips PCR Machines Methods:
1. Set-up PCR mixture: Students will set-up the PCR mixture (2 x 50 µl) including EGFP-specific oligonucleotides. To one tube, they will add the pEGFP-C1 template DNA. In parallel, they will also set up a negative control tube with water substituted for the DNA template. (Time = 30 minutes)
A) Calculate the amounts of ingredients you will need for each 50µl PCR reaction.
EGFP Rx PCR Components Negative Control Rx
1 µl EGFP Template 0 µl
5 µl 10X buffer 5 µl
1 µl dNTPs 1 µl
1 µl 5’ oligonucleotide 1 µl
1 µl 3’ oligonucleotide 1 µl
1 µl Taq polymerase 1 µl
40 µl water 41 µl
50 µl Total Volume 50 µl
B) Compose a “Master Mix’ including all of the components except for the template and an equivolume of water for the negative control. (Ex: if utilizing 1 µl of DNA template, leave out 1 µl of water from the master mix). Keep the mixtures on ice and add the taq polymerase last.
C) Divide 49 µl of the Master Mix into two PCR reaction tubes on ice.
D) Add 1µl of pEGFP-C1 DNA to one tube. Add 1µl of water to the negative control tube. Close tubes and label accordingly. Keep samples on ice.
2. PCR Amplification: Students will place tubes in the PCR machine, carefully recording their position. They will observe the Lab Assistant set-up the program. The Lab Assistant will review each of the steps of PCR amplification while he/she sets-up the program. Students will be required to record the parameters for their lab report.
The Lab Assistant will begin the amplification. (Time = 20 minutes for set-up and explanation; amplification will take approximately 2-2.5 hours).
Exercise 2: Confirmation of PCR Amplification and Initialization of Expression Plasmid Construction
Objective:
Students will use gel electrophoresis to confirm PCR amplification and begin preparation for expression plasmid construction.
Materials:
Agarose Gel electrophoresis apparatus (trays, combs, boats, power supplies) TAE Buffer (1X)
100 bp DNA Ladder Loading dye
Syber Green I dye
MultiImage Light Cabinet Pipettemen
Pipette tips Eppendorf Tubes Methods:
Preparation of agarose gels:
1.Assemble the minigel electrophoresis apparatus according manufacturer’s instructions.
The BRL Baby Gel is representative of the many gel systems available and comes with a removable through in which tp form the gel. Tape the ends of the trough and position the trough in the apparatus.
2.Melt 0.2g of agarose in 25ml of 1X TAE buffer, using microwave oven or a Bunsen burner, and equilibrate to 45°C in a water bath.
3.fill the trough with agarose and immediately insert the comb. Act quickly to remove any bubbles by touching them with a Pasteur pipet.
4.Allow the gel to solidify at room temperature for 20 minutes. Gently remove the comb.
5.Remove the tape from the trough and fill the reservoirs with 1X TAE buffer to just cover the gel.
Rrunning of the gels:
1. Students will observe the Lab Assistant construct agarose gels for visualization of their amplified PCR products. (Time = 20 minutes)
2. Students will mix 2µl of each of their PCR mixtures with 2µl of diluted loading dye in separate tubes. The remainder of the PCR mixtures will be stored at 4°C for use during expression plasmid construction. (Time = 20 minutes)
A)Set-up two eppendorf tubes in a rack at your bench. Label one tube “EGFP” and the other “-“.
B)Add 2µl of diluted loading dye to each tube.
D)Add 2µl of the negative control PCR reaction to the tube labeled “-”. Mix gently by pipetting.
3. Students will carefully load each of their samples onto the agarose gel. Students must record in which lanes of the gel their samples are loaded. The Lab Assistant will load 100 bp DNA ladder as a marker lane. The gel will undergo electrophoresis for approximately 30-45 minutes. (Time = 1 hour 15 minutes).
4. Students will observe as the Lab Assistant stains the gels and move the gels to MultiImage Light Cabinet for visualization and recording of electrophoresed PCR products. (Time = 20 minutes)
Exercise 3: Restriction Enzyme Digestion
Objective:
In order to get the favorable DNA fragments, Students have to cut the PCR product and pET30b(+) expression plasmid using the same selected restriction enzyme.
Materials:
pET30b(+) expression plasmid ---map on next page EGFP PCR product
restriction enzyme buffer
restriction enzyme : NdeI and EcoRI Eppendorf Tubes
Heating plate Methods:
Vector preparation:
1. Assemble the following components in a microcentrifuge tube:
Component Volume
pET30b(+) plasmid 30 µl Restriction enzyme buffer (10x) 5 µl
Restriction enzyme: NdeI 2 µl Restriction enzyme: EcoRI 2µl
water 11 µl
Total Volume 50.0 µl
2. Incubate at the appropriate temperature (usually 37°C) for 6~16 h.
3. Add 7 µl mixtures(loading dye : Syber Green I:water =4:3:3) to the reaction and load the entire sample into a large well on a 1% agarose gel. Run the gel far enough to separate the linear plasmid from nicked and supercoiled species. It is useful to run uncut vector DNA in an adjacent lane to help distinguish undigested from linearized plasmid DNA.
4. Visualize the DNA band with a long wave UV light source and excise the band from the gel using a clean razor blade. Minimize exposure to the light source, which can cause nicks and double strand breaks in the DNA.
5. Recover the DNA from the gel.The Gel extraction DNA Kit is ideal for this application.
Resuspend the final product in a total volume of 35 µl (usually about 50 ng/µl DNA).
Insert preparation:
1. Assemble the following components in a microcentrifuge tube:
Component Volume EGFP PCR product 30 µl Restriction enzyme buffer (10x) 5 µl
Restriction enzyme: NdeI 2 µl Restriction enzyme: EcoRI 2µl
water 11 µl
Total Volume 50.0 µl
2. Incubate at the appropriate temperature (usually 37°C) for 12~16 h.
3. Add 7 µl mixtures(loading dye and Sybr Green I) to the reaction and load the entire sample into a large well (0.5–1.0 cm wide) on a 1% agarose gel. Run the gel far enough to separate the linear plasmid from nicked and supercoiled species. It is useful to run uncut vector DNA in an adjacent lane to help distinguish undigested from linearized plasmid DNA.
4. Visualize the DNA band with a long wave UV light source and excise the band from the gel using a clean razor blade. Minimize exposure to the light source, which can cause nicks and double strand breaks in the DNA.
5. Recover the DNA from the gel.The Gel extraction DNA Kit is ideal for this application.
Resuspend the final product in a total volume of 35 µl (usually about 50 ng/µl DNA).
Exercise 4: Expression Plasmid Construction
Objective:
Students will ligate the PCR-amplified EGFP product with the pET30b(+) expression plasmid. Competent bacteria will be transformed with the plasmid and plated on LB-ampicillin plates for bacterial growth.
Materials:
pET30b(+) expression plasmid ligation buffer
ligase
LB-agar plates Kanamycin
Spreaders Rotating Plating platforms Top10 competent bacteria
Eppendorf Tubes Ice/ Ice buckets LB Broth Bacterial shaker Eppendorf centrifuges Methods:
1. Students will be supplied with a sample of the pET30b(+) expression plasmid. This plasmid is already digested for PCR fragment insertion and does not require that PCR fragments need to be cleaned up prior to ligation.
2. Ligation of EGFP PCR product with pET30b(+) plasmid: Students will prepare a ligation mix (Total volume = 20 µl) including the pET30b(+) (4 µl) and the EGFP PCR product (12 µl). Add ligase as the last component. Ligation requires 6h at 16°C.
(Time = 7 h)
Component Volume
pET30b(+) plasmid 4 µl
EGFP insert 12 µl
Ligation buffer (10x) 2 µl
ATP 1µl
ligase 1 µl
Total Volume 20.0 µl 3. Preparation of LB-Kan Agar plates:
A) Take two LB agar plates to the plate preparation station.
B) Using the pipetteman, add Kan to the surface of your plate. The Kan stock is 30µg/µl.Therefore, add 20µl of the antibiotic to each plate. Be careful to not touch
spreading the ampicillin on each agar plate.
D) Touch the spreader on the agar away from the ampicillin droplet. This ensures that the spreader is no longer too hot. Turn the plate clockwise and you move the spreader back and forth over the surface of the agar to spread the antibiotic evenly.
Repeat for steps C and D for the second plate.
E) Store your plates at 4°C for later use.
4. Bacterial Transformation:
Students will be supplied with a sample of Top10 competent bacteria. Bacteria will be transformed with the ligation mix using the heat-shock method (30 minutes on ice;
120 seconds at 42°C; 2 minutes on ice; add 0.5~1ml of LB broth; 1 hour at 37°C in bacterial shaker). (Time= 1 hour 50 minutes)
A)Pick up a sample of Top10 competent bacteria for bacterial transformation of your pET30b(+)-EGFP plasmid. Keep all components on ice.
B)Add 20µl of the pET30b(+)-EGFP ligation mix prepared in Step 2 to the tube of Top10 competent bacteria. Place this mixture on ice for 30 minutes.
C)After 30 minutes have expired, place the tube containing the mixture in a 42°C water bath for 120 seconds.
D)After 120 seconds, place the mixture back on ice for 2 minutes.
E) Add 0.5~1ml of LB broth to the mixture and place the tube at 37°C in the bacterial shaker for 1 hour.
F)Retrieve the LB-Kan plated from the 4°C refrigerator to warm up to room temperature at your bench.
5. Following growth for 1 hour, Students will spread 100µl and 200µl on the two LB-Kan plates. Plates will be incubated at 37°C over night. (Time = 10 minutes).
A) Label on agar plate “100- pET30b(+)-EGFP ”, your name and date. Label the other
“200- pET30b(+)-EGFP”, your name and date.
B) Pipette 100µl onto the surface of the 100- pET30b(+)-EGFP agar plate and 200 µl on the 200- pET30b(+)-EGFP plate.
C) Move to the plate preparation station.
D)Dip the glass spreader in ethanol and flame using the bunsen burner provided.
Remove the spreader from the flame and count to 20 or time 20 seconds prior to spreading the bacteria on the agar plate.
E)Touch the spreader on the agar away from the bacterial droplet. This ensures that the spreader is no longer too hot. Turn the plate clockwise and you move the spreader back and forth over the surface of the agar to spread the bacteria evenly.
Repeat for steps D and E for the second plate. Store your plates in the bacterial incubator at 37°C for next laboratory session.
Exercise 5: Transformation of Escherichia coli by plasmid DNA
Objective:
The most common method of bacterial transformation utilizes high levels of calcium chloride to facilitate the entry of plasmid DNA vectors into Escherichis coli by causing structural alterations in the bacteria; cell wall. In general, there are three basic steps in the introduction of plasmid DNA into cells: (1) preparation of competent cells, (2) transformation of the competent cells, and (3) selection of transformants; Following the preparation of competent cells, transformation is induced by destabilization of the lipids in the cell envelope through heat-shock treatment. The various parameters and constraints involved in attaining good transformation efficiency differ between cell lines and vectors.
Factors, such as plasmid size, DNA configuration (closed, linear, or nicked), and antibiotic/marker selection, may all influence the outcome of a transformation experiment.
Materials:
100mM Calcium chloride solution Centrifuge tubes
Escherichia coli Top10
LB agar plate with 30µg/ml kanamycin LB broth
TE buffer Eppendorf Tubes
50% (v/v) glycerol stock solution Methods:
Preparation of competent cells:
1.Aseptically prepare a 3-ml overnight culture of E.coli Top10 in LB broth in test tube on a shaker at 37
o
C.
2.The next day prepare a subculture by transferring 1ml of the overnight cells into a 250ml flask containing 30ml of LB broth. Grow the cells with vigorous shaking for 1 hour.
3.After 1 hour, remove the cell into a prechilled, sterile centrifuge tube. Chill for 10 minutes in an ice bath.
4.Collect the cells by centrifugation at 5000rpm for 5 minutes in 1 centrifuge at 4
o
C.
5.Carefully discard the supernatant and gently resuspend the pellet in 20ml of sterile, ice-cold 100mM CaCl2 solution in an ice bath for 30 minutes.
6.Centrifuge at 5000rpm for 5 minutes at 4
o
C.
(Meanwhile, prepare 60 l glycerol into 20 microtubes)
7.Discard the supernatant and gently resuspend the pellet in 4ml of ice-cold CaCl2
solution.
8. Add 140 µl of the above solution into #6 glycerol tubes.
Transformation of the competent cells:
1.Remove the cells from refrigeration, gently resuspend, and add the plasmid DNA. Mix and place on ice for 30 minutes.
2.Heat shock the cells by incubating the tube in a 42
o
C heating plate for 2 minutes.
3.Add 1.0 ml of LB broth to the tube and incubate at 37 oC for 1 hours, which occasional gentle mixing, to allow for expression of the selective marker.
4.Add 100~200µl of transformed culture to LB agar plate with selective pressure and incubate at 37 oC overnight.
Exercise 6: Analysis of Bacterial Growth Plates and Selection of Clones
Objective:
Students will select bacterial colonies that express the EGFP protein. PCR will be used to confirm bacterial clones containing the gene encoding for EGFP. A selected EGFP-positive bacterial clone will be expanded for DNA isolation in the next lab sessions.
Materials:
Pipettemen Yellow tips Eppendorf Tubes PCR reaction mix Taq polymerase
EGFP oligonucleotides pEGFP-C1 plasmid PCR Machines LB-kan Broth 15 ml Culture tubes Serological pipettes Bacterial Shaker.
Methods:
1. Colony Selection: Students will select 6 colonies from each plate using a toothpick, placing each colony on new LB+Kan plate.(Time = 10 minutes)
A) Using a sterile toothpick, pick 12 colonies and streak or patch on LB+Kan plate.Touch the tip to one of the circled colonies. Try not to stab the agar.
B) Incubate the plates at 37°C for 4~6 h.
2. Bacterial Growth: The student will place their plate in the orbital shaker at 37°C for 4~6 hour. (Time = 4~6 h).
3. PCR Analysis: After 4~6 hours of growth, the students will set up 14 PCR reactions(20µl/ tube) containing the EGFP oligonucleotides. Two tubes will receive 1µl of the original plasmid pEGFP-C1 as a positive control and water as a negative control respectively. (Time = 30 minutes).
A) Prepare the following 300 µl PCR Master Mix devoid of template to create 14 PCR reactions:
Master Mix PCR Components 0 µl EGFP Template 30 µl 10X buffer
3 µl dNTPs
3 µl 5’ oligonucleotide 3 µl 3’ oligonucleotide 3 µl Taq polymerase
258 µl water
300 µl Total Volume B) Label 14 PCR reaction tubes: 1~12, + and -.
C) Pipette 20 µl of master mix into each PCR reaction tube.
D) Add 1 µl of sterile water to the “-“ control tube and close the tube.
E) Using a sterile toothpick to pick up #1 colony to the “1“ tube and close the tube.
F) Using a sterile toothpick to pick up #1 colony to the “2“ tube and close the tube.
G) Add 1 µl of the original pEGFP-C1 to the “+“ tube and close the tube.
H) Transport tubes to the PCR machine.
4. The Lab Assistant will set-up the PCR program for bacterial screening. The students will record the program for their report and load the machine with their tubes.. (Time = 2 hours).
5.Analysis of PCR Screening: Students will retrieve their PCR samples from the refrigerator. Samples will be mixed with loading dye and loaded onto an agarose gel prepared by the Lab Assistant. The gel will undergo electrophoresis for 30 minutes and subsequently stained with Syber Green I dye. The gel will be analyzed using the Gel Documentation System. Each student will receive a print-out of their samples that must be labeled and included with their report. (Time = 60 minutes).
A)Retrieve your PCR Samples from the refrigerator.
B)Add 1 µl of diluted loading dye to each PCR reaction. Change your pipette tip after the addition of dye to each tube.
C)Load your samples on the agarose gel prepared by the lab assistant. Carefully record the position of your samples.
D)The gel will undergo electrophoresis for 30 minutes at 120 volts.
E)After 30 minutes, the gel will be visualized using the gel documentation system.
6. After the PCR screening, the students will select EGFP+ bacterial colony that fluoresced in the presence of the fluorescent light source. This colony will be added to 4 ml of LB-Kan broth in a 15 ml culture tube and placed in the bacterial shaker overnight. (Time = 15 minutes). This culture will be used in the next lab session for DNA isolation.
A)Add 5 ml of LB-Kan broth to a 15 ml bacterial culture tube.
B)Take the culture tube and one of your EGFP+ bacterial colony plates to the Culture preparation Station. .
C)Touch the sterile stick to to select a colony that was previously identified as EGFP+. D)Take the stick with colony and place it in the culture tube with the 3ml of LB-Kan
broth. Stir the broth with the stick briefly.
E) Place the culture at 37°C.
Exercise 7: DNA Isolation
Objectives:
The student will isolate plasmid DNA from the overnight bacterial cultures. Students will prepare a restriction digest of the isolated plasmid DNA to confirm proper vector construction.
Materials:
Agarose
Gel electrophoresis apparatus (trays, combs, boats, power supplies) TAE Buffer (1X)
100 bp DNA Ladder Loading dye
Sybr Green I dye Pipettemen Pipette tips Eppendorf Tubes
Qiagen Mini Prep Plasmid DNA Kits Methods:
1. Bacterial Plasmid DNA Isolation: Students will retrieve the overnight bacterial cultures.
Students will use a Qiagen Mini Prep DNA kit to isolate plasmid DNA. (Time = 60 minutes) according the manufactures guidelines.
A)Centrifuge 3 ml of bacterial culture in an 1.5 ml tube. Resuspend pelleted bacterial cells in 250 µl of Buffer P1 and transfer to a microfuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
B)Add 250 µl of Buffer P2 and gently invert the tube 4–6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
C)Add 350 µl of Buffer N3 and invert the tube immediately but gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
D)Centrifuge for 10 min. A compact white pellet will form. During centrifugation, place a QIAprep spin column in a 2 ml collection tube.
E)Apply the supernatants from step 4 to the QIAprep column by decanting or pipetting.
F)Centrifuge 30–60 sec. Discard the flow-through.
G)Wash QIAprep spin column by adding 0.75 ml of Buffer PE and centrifuging 30–60 sec.
H)Place QIAprep column in a clean 1.5 ml microfuge tube. To elute DNA, add 50 µl of Buffer EB (10 mM Tris·Cl, pH 8.5) or H2O to the center of the QIAprep column, let stand for 1 min, and centrifuge for 1 min.
