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.

Genome-wide searches conducted in recent years have uncovered ~70 sRNAs encoded by the chromosome of the enterobacterium Escherichia coli alone.

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.

Introduction

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

ResultsMiniprep 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

ResultsPCR 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

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 : ompA=1:4

4 16 0

100

8

Plasmid :

ompA-long-

=1:5

3 15 2 1

Plasmid : ompF=1:10

1 10 8 14

2

Plasmid : ompA=1:10

1 10 9 100

150

5

13

Plasmid :

ompA-long-

=1:10

1 10 9 100

150

6

9

Plasmid : ompF=1:10

1 10 9 100

150

20

14

plasmid 1 0 19 100

150

6

18

3

plasmid :

ompA-long-=1:15

1 15 4 50

100

9

1

Plasmid :

ompA-long-=1:10

1 10 9 50

100

3

4

Plasmid :

ompA-long-=1:5

2 10 8 50

100

3

5

plasmid 1 0 19 50

100

3

5

Results

Results

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

Colony PCR for the first transformation

ResultsColony 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

ResultsColony 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

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.

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.

Thanks!