Transcript E. coli strain

Creating an RNAi feeding vector
How does ligation into L4440 work?
Creating an RNAi feeding vector
Taq preferentially adds an extra “A” to the 3’ end of PCR products
Creating an RNAi feeding vector
Taq preferentially adds an extra “A” to the 3’ end of PCR products
A
A
Creating an RNAi feeding vector
L4440
Taq will add a 3’ T
to blunt-cut L4440 vector if
there is only dTTP in the
reaction buffer
Creating an RNAi feeding vector
L4440
Taq will add a 3’ T
to blunt-cut L4440 vector if
there is only dTTP in the
reaction buffer
Creating an RNAi feeding vector
A
A
L4440
Ligate PCR product
into T-tailed vector
L4440
Creating an RNAi feeding vector
Taq preferentially adds an extra “A” to the 3’ end of PCR products
A
A
L4440
Ligate PCR product
into T-tailed vector
L4440
Creating an RNAi feeding vector
Taq preferentially adds an extra “A” to the 3’ end of PCR products
A
A
L4440
Ligate PCR product
into T-tailed vector
L4440
This is a very efficient and useful process:
•Works for any PCR product
•The extra T on the vector inhibits vector self-ligation
How does the vector induce RNAi?
•Allows E. coli to produces double-stranded RNA
•Requires a specific strain of E. coli
The RNAi feeding vector has two T7 RNA polymerase
promoters
•T7 RNA polymerase is a viral polymerase with
its own promoter sequence
•This allows transcription of the two strands of
RNA from the insert between the promoters
E. coli strain HT115(DE3) has a modified lac promoter
controlling the transcription of T7 RNA polymerase
T7 RNA polymerase
T7 RNA polymerase
Regulation of Lac Operon transcription
IPTG also induces
When IPTG is added T7 RNA polymerase is expressed
T7 RNA polymerase