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GFP pJV861-9 cloning

GFP pJV861-9 cloning. Name : Yao Shi Supervisor : Professor E. Gerhart H. Wagner. Introduction. Antisense small RNAs(sRNAs) are a class of regulators of gene expression that act on target RNAs via sequence complementarity.

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GFP pJV861-9 cloning

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  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 sequencing

  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

  8. Results

  9. 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

  10. 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

  11. Results Colony PCR for the third transformation 500bp 400bp 300bp 200bp 100bp 1 2 3 4 5 6 7 8 9 1011121314151617181920212223242526272829 1-25 : from ompA-long- colony 26,27 : from original pJV861-9 28 : no template 29 : marker

  12. 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.

  13. 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.

  14. Thanks!

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