Genetic Transformation in Escherichia coli K12

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1 Genetic Transformation in Escherichia coli K12
SHARON D. COSLOY AND MICHIO OISHI 1973 Group 5 劉穎璞 林思慧 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。 2018/12/7 Principles of Gene Manipulation

2 INTRODUCTION 2018/12/7 Principles of Gene Manipulation

3 Transformation S型 R型 Frederick Griffith 1879–1941
Streptococcus pneumoniae 2018/12/7 Principles of Gene Manipulation

4 Escherichia coli K12 double-stranded circular DNA ATP-dependent DNase
Transformation 時，通常是 supercoil 。 但怕可能會有nick，所以還是會把DNase去掉。 ATP-dependent DNase double-stranded circular DNA linear DNA does not attack attack Transformation 時，若是利用linear DNA，因為DNase會攻擊linear DNA，所以一般把DNase去掉。 2018/12/7 Principles of Gene Manipulation

5 MATERIALS AND METHODS 2018/12/7 Principles of Gene Manipulation

6 Table1. Bacterial strains used in this study
K12有許多不同種的菌株，作者特別挑這幾種的原因主要是因為他們的某些基因突變，故有利於做菌落篩選及比較。 2018/12/7 Principles of Gene Manipulation

7 符號的意義 Ex: 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) 符號 意義 end
endonuclease I Leu Leucine His Histine ara 阿拉伯糖 Rec B、Rec C ATP-depentaent DNase 2018/12/7 Principles of Gene Manipulation

8 Isolation of Transforming DNA
NaCl - EDTA Tris HCl-SDS centrifugation room temperature gently shaken for 20 min phenol centrifugation 2018/12/7 Principles of Gene Manipulation

9 Withdraw the aqueous phase two volumes of 95% ethyl alcohol
saline-citrate (contained EDTA). 37° 30 min RNase phenol ethyl alcohol Pure DNA DNA dissolve 2018/12/7 Principles of Gene Manipulation

10 Transformation overnight 37° transferred to fresh medium
incubated with rotatory shaking (the cell density reached 3 X 108/ml.) P medium (required amino acids + vitamin B1) 0° 20 min Chilled (0~2°) centrifuged Treatment with CaCl2 centrifuged 2018/12/7 Principles of Gene Manipulation

11 + CaCl2 transferred cell suspension DNA + CaCl2 45 min at 0°
Terminated at 0°. + DNase 2018/12/7 Principles of Gene Manipulation

12 我們之後篩選colonies是利用產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。
minimal medium required (nonselective) amino acids vitamins. 我們之後篩選colonies是利用產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 36~48 hr at 37° 2018/12/7 Principles of Gene Manipulation

13 RESULT 2018/12/7 Principles of Gene Manipulation

14 various concentration
Transformation various concentration (table 2) various treatments (table 3) various sources (table 4) Linkage (table 5、6) 2018/12/7 Principles of Gene Manipulation

15 Table2. Transformation with various concentration of DNA
←Donor DNA ←Recipient transformants produced ∝ the amount of DNA These results indicate that transformation events are not limited to a few particular genetic markers but occur generally for the available markers located at several places on the E. coli chromosome, thus suggesting that this system is similar to the other known bacterial transformation systems. 2018/12/7 Principles of Gene Manipulation

16 Table3. 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2.8X108，(B)則是2.1X108，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20分鐘 biologically active component in the DNA preparation is DNA only double-stranded DNA is active for the transformation 反應15分鐘 2018/12/7 Principles of Gene Manipulation

17 Table4. Transformation with DNA various sources
產生誤差可能的原因： 人為操作上的誤差(因為當時的技術不是很成熟)。 細菌可能自己產生突變(因為機率不大，所以只有一兩個)。 2018/12/7 Principles of Gene Manipulation

18 Table5. Evidence for linkage by transformation
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) 2018/12/7 Principles of Gene Manipulation

19 結果 Donor DNA (HfrC6) Recipient cell (JC7623)
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。 培養完之後再隨機抽50個colonies 做分析。 結果 結論： 分析後發現，ara+成功轉形的機率是40%，而his+為0%。所以他推測leu與ara間的距離較近，與his較遠。 ⇒linkage可能會有影響。 These results indicate that physical breakage of the DNA molecules causes loss of the genetic linkage originally observed, and further support the interpretation that the biologically active fraction in the DNA preparation must be DNA itself. 因為過去還不知道甚麼樣的DNA容易進行transformation，故兩種都做。 2018/12/7 Principles of Gene Manipulation

20 Table6. Cotransformation of closely linked negative genetic markers
想法：剛剛是倆倆比較，現在三種同時比較。 做法：cotransformation 2018/12/7 Principles of Gene Manipulation

21 結果 MO617 MO618 MO619 之後再用medium培養，用 leu+ 來進行篩選。
Donor DNA (leu+,ara-,his-) (leu+,ara+,his-) Recipient cell (leu-,ara+,his+) Genotype (leu+,ara-,his-) 預期 MO617 MO618 MO619 之後再用medium培養，用 leu+ 來進行篩選。 培養完之後再隨機抽50個colonies 做分析。 結果 Leu+ transformants were selected and tested to determine if they had incorporated the donor's aramarker. 結論： 分析後發現，在ara-的部分，MO617成功轉形且機率是34%(與表5推論相同)，而MO618為0%(符合，因為它是ara+)。在his-部分，兩者皆為零(與表5推論符合，因leu離his較遠。) ⇒linkage 會有影響。 2018/12/7 Principles of Gene Manipulation

22 DISCUSSION 2018/12/7 Principles of Gene Manipulation

23 Discussion It’s similar to other bacterial transformation system.
other possibilities that could explain these results : DNA of some undetected phages or plasmids DNA of a particular structure 2018/12/7 Principles of Gene Manipulation

24 Require high concentrations of DNA. Why ?
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. 2018/12/7 Principles of Gene Manipulation

25 Thanks for your listening !!
2018/12/7 Principles of Gene Manipulation