GFP pJV861-9 cloning

1
GFP pJV861-9 cloning

• Name Yao Shi
• Supervisor Professor
• E. Gerhart H. Wagner

2
Introduction

• Antisense small RNAs(sRNAs) are a class of
  regulators of gene expression that act on target
  RNAs via sequence complementarity.
• Genome-wide searches conducted in recent years
  have uncovered 70 sRNAs encoded by the
  chromosome of the enterobacterium Escherichia
  coli alone.

3
Introduction

• Johan Reimegard made a Genome-wide search of the
  possible sRNA targets in Escherichia coli and
  selected some of them to be tested and ompA,
  ompA-long-, ompF are three of them.

4
Introduction
5
Methods

• miniprep of pJV861-9
  PCR amplification of the target
• 
  mRNA fragment
  ompA,
• 
  ompA-long-
  ,ompF
• cleavage of the pJV861-9
  cleavage of the amplified fragments
• gel purification
  gel
  purification
• 
  ligation
• 
  transformation
• 
  colony PCR
• miniprep of
  pJV861-9 with insertion
• PCR amplify
  the insertion fragment
• 
  gel purification

6
Results

• Miniprep and cleavage of pJV861-9

10000bp 4000bp 2000bp 1000bp 500bp
1 2 3 4
5
1 marker 2 not cleaved pJV861-9 3,4,5 cleaved
pJv861-9
7
Results

• PCR amplification of the target mRNA fragments

400bp 300bp 200bp 100bp
1 2 3 4 5 6 7
8 9 10 11 12 13
1,2,3,4 amplified ompA fragment 5,6,7,8
amplified ompA-long- fragment 9,10,11,12
amplified ompF fragment 13 marker
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Results
Number of transformation Ratio of plasmid to fragment ( v v) Plasmid (µl) Fragment (µl) dH2O (µl) Volume spread on plate (µl) number of colonies
1 Plasmid ompA14 4 16 0 100 8
1 Plasmid ompA-long- 15 3 15 2 100 1
1 Plasmid ompF110 1 10 8 100 14
2 Plasmid ompA110 1 10 9 100 150 5 13
2 Plasmid ompA-long- 110 1 10 9 100 150 6 9
2 Plasmid ompF110 1 10 9 100 150 20 14
2 plasmid 1 0 19 100 150 6 18
3 plasmid ompA-long-115 1 15 4 50 100 9 1
3 Plasmid ompA-long-110 1 10 9 50 100 3 4
3 Plasmid ompA-long-15 2 10 8 50 100 3 5
3 plasmid 1 0 19 50 100 3 5
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Results
Colony PCR for the first transformation
400bp 300bp 200bp 100bp
1 2 3 4 5 6 7 8 9 10 11 12
13 14 15 16 17 18 19 20 21 22 23 24 25 26
1-8 from ompA colony 9 from ompA-long-
colony 10-23 from ompF colony 24 from original
pJV861-9 25 no template 26 marker
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Results

• Colony PCR for the second transformation

400bp 300bp 200bp 100bp
1 2 3 4 5 6 7 8 9 10 11 12 1314 15 16 17
18 19 202122 23 242526 2728 29 30
1-6 from ompA conlony 7-21 from ompA-long-
colony 22-26 from ompF colony 27,28 from
original pJV861-9 plasmid 29 no template 30
marker
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Results

• Colony PCR for the third transformation

500bp 400bp 300bp 200bp 100bp
1 2 3 4 5 6 7 8 9 1011121314151617181920
212223242526272829
1-25 from ompA-long- colony 26,27 from
original pJV861-9 28 no template 29 marker
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Summary

• After a flow- minipreparation of the plasmid,
  PCR amplification the target mRNA fragments,
  cleavage of the plasmid and fragments, ligation
  and transformation, I finally got some colonies
  with ompA and ompF insertion.
• Only after sequencing, could I make a
  conclusion that I got the colonies with the right
  insertion.

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Future plans

• Do more construction of plasmids with predicted
  target mRNA fragments insertion.
• Co-transform the GFP plasmid pJV861-9 with the
  target mRNA fragment insertion and the plasmid
  with antisense small RNA insertion to see the GFP
  expression.

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Thanks!