Genetic Transformation in Escherichia coli K 12 SHARON

INTRODUCTION 2021/9/3 2 Principles of Gene Manipulation

Transformation S型 R型 Frederick Griffith 1879– 1941 Streptococcus pneumoniae 2021/9/3 3 Principles of Gene

Escherichia coli K 12 Transformation 時，通常是 supercoil 。但怕可能會有nick，所以還是會把 DNase去掉。 does not attack double-stranded

MATERIALS AND METHODS 2021/9/3 5 Principles of Gene Manipulation

符號的意義 ß ß 意義 endonuclease I Leucine Histine ara 阿拉伯糖 Rec B、Rec C ATP-depentaent

Isolation of Transforming DNA 42° 2 -5 min 1. Na. Cl - EDTA 2.

Withdraw the aqueous phase two volumes of 95% ethyl alcohol saline-citrate (contained EDTA). 37°

Transformation overnight 37° 1. transferred to fresh medium 2. incubated with rotatory shaking (the

+ Ca. Cl 2 transferred cell suspension DNA + Ca. Cl 2 45 min

1. minimal medium 2. required (nonselective) amino acids 3. vitamins. 我們之後篩選colonies是利用�生胺基酸的基因是否有表達來篩選，所以我們只加那些非篩選用的胺基酸，避免發生錯誤。

RESULT 2021/9/3 13 Principles of Gene Manipulation

various treatments (table 3) various concentratio n (table 2) 2021/9/3 various sources (table 4)

Table 2. Transformation with various concentration of DNA ←Donor DNA ←Recipient 2021/9/3 15 Principles

Table 3. Effect of various treatments of DNA on the transforming activity ←Donor DNA

Table 5. Evidence for linkage by transformation ß ß ß 2021/9/3 問題：Linkage 會不會影響transformation 的效率(成功與否)?

Recipien t cell (JC 7623 ) Donor DNA (Hfr. C 6) 培養完之後再隨機抽 50個 colonies

Table 6. Cotransformation of closely linked negative genetic markers ß 問題：再次驗證table 5的推論? ß 想法：剛剛是倆倆比較，現在三種同時比較。

Donor DNA (leu+, ara-, his) (leu+, ara+, his) 培養完之後再隨機抽 50個 colonies 做分析。 2021/9/3 MO

DISCUSSION 2021/9/3 22 Principles of Gene Manipulation

Discussion ß ß It’s similar to other bacterial transformation system. other possibilities that could

ß Require high concentrations of DNA. Why ? Þ ß 2021/9/3 E. coli cells

Thanks for your listening !! 2021/9/3 25 Principles of Gene Manipulation

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Genetic Transformation in Escherichia coli K 12 SHARON D. COSLOY AND MICHIO OISHI 1973 Group 5 992312 劉穎璞 992334 林思慧 992339 黃珮瑄 [註] 這是修改過後的，中文部分是當初覺得沒講清楚以及同學提問的。 2021/9/3 1 Principles of Gene Manipulation

INTRODUCTION 2021/9/3 2 Principles of Gene Manipulation

Transformation S型 R型 Frederick Griffith 1879– 1941 Streptococcus pneumoniae 2021/9/3 3 Principles of Gene Manipulation

Escherichia coli K 12 Transformation 時，通常是 supercoil 。但怕可能會有nick，所以還是會把 DNase去掉。 does not attack double-stranded circular DNA ATP-dependent DNase attack 2021/9/3 Transformation 時，若是利用linear DNA，因為DNase會攻擊linear DNA，所以一般把DNase去掉。 4 linear DNA Principles of Gene Manipulation

MATERIALS AND METHODS 2021/9/3 5 Principles of Gene Manipulation

符號的意義 ß ß 意義 endonuclease I Leucine Histine ara 阿拉伯糖 Rec B、Rec C ATP-depentaent DNase Ex: Þ Þ 2021/9/3 符號 Leu+：該基因有表達。 Leu(或Leu-)：該基因沒有表達。(可能是突變、缺失等等) 7 Principles of Gene Manipulation

Isolation of Transforming DNA 42° 2 -5 min 1. Na. Cl - EDTA 2. Tris HCl-SDS centrifugation room temperature gently shaken for 20 min phenol 2021/9/3 centrifugation 8 Principles of Gene Manipulation

Withdraw the aqueous phase two volumes of 95% ethyl alcohol saline-citrate (contained EDTA). 37° 30 min RNase phenol Pure DNA dissolve 2021/9/3 ethyl alcohol 9 Principles of Gene Manipulation

Transformation overnight 37° 1. transferred to fresh medium 2. incubated with rotatory shaking (the cell density reached 3 X 108/ml. ) P medium (required amino acids + vitamin B 1) Chilled (0~2°) 2021/9/3 centrifuged 0° 20 min Treatment with Ca. Cl 2 10 centrifuged Principles of Gene Manipulation

+ Ca. Cl 2 transferred cell suspension DNA + Ca. Cl 2 45 min at 0° 2021/9/3 10 min at 37°. Terminated at 0°. 11 + DNase Principles of Gene Manipulation

1. minimal medium 2. required (nonselective) amino acids 3. vitamins. 我們之後篩選colonies是利用�生胺基酸的基因是否有表達來篩選，所以我們只加那些非篩選用的胺基酸，避免發生錯誤。 Resul t 36~48 hr at 37° 2021/9/3 12 Principles of Gene Manipulation

RESULT 2021/9/3 13 Principles of Gene Manipulation

various treatments (table 3) various concentratio n (table 2) 2021/9/3 various sources (table 4) Linkage (table 5、6) Transformatio n 14 Principles of Gene Manipulation

Table 2. Transformation with various concentration of DNA ←Donor DNA ←Recipient 2021/9/3 15 Principles of Gene Manipulation

Table 3. Effect of various treatments of DNA on the transforming activity ←Donor DNA ←Recipient 分成兩部份的原因 paper中並沒有講，但他有提到(A)的細胞數是 2. 8 X 108， (B)則是 2. 1 X 108，所以我們猜測可能是兩個不同的人或是不同天做實驗。反應20分鐘反應15分鐘 2021/9/3 16 Principles of Gene Manipulation

Table 5. Evidence for linkage by transformation ß ß ß 2021/9/3 問題：Linkage 會不會影響transformation 的效率(成功與否)? 想法： Linkage → marker 之間的距離作法：cotransformation (同時轉進 2個或 2 個以上基因稱之) 18 Principles of Gene Manipulation

Recipien t cell (JC 7623 ) Donor DNA (Hfr. C 6) 培養完之後再隨機抽 50個 colonies 做分析。結果 Genotype (leu+, ara+, his +) 預期之後再用medium培養，分別用表中 selected marker 來進行篩選。因為過去還不知道甚麼樣的DNA容易進行transformation，故兩種都做。 2021/9/3 19 Principles of Gene Manipulation

Table 6. Cotransformation of closely linked negative genetic markers ß 問題：再次驗證table 5的推論? ß 想法：剛剛是倆倆比較，現在三種同時比較。 ß 做法：cotransformation 2021/9/3 20 Principles of Gene Manipulation

Donor DNA (leu+, ara-, his) (leu+, ara+, his) 培養完之後再隨機抽 50個 colonies 做分析。 2021/9/3 MO 617 Genotype + Recipient cell (leu, ara+, his+) MO 619 結果 21 (leu , ara , his) (leu+, ara+, his) MO 618 預期之後再用medium培養，用 leu+ 來進行篩選。 Principles of Gene Manipulation

DISCUSSION 2021/9/3 22 Principles of Gene Manipulation

Discussion ß ß It’s similar to other bacterial transformation system. other possibilities that could explain these results : Þ Þ 2021/9/3 DNA of some undetected phages or plasmids DNA of a particular structure 23 Principles of Gene Manipulation

ß Require high concentrations of DNA. Why ? Þ ß 2021/9/3 E. coli cells inherently do not have an active uptake system for incorporating DNA. The potential for using this organism as a model to study the introduction and integration of genetic information from various sources. 24 Principles of Gene Manipulation

Thanks for your listening !! 2021/9/3 25 Principles of Gene Manipulation