Part:BBa_K3815013

Part of the EPH1 gene of Ipomoea nil to synthesize dsRNA for RNAi
This is a section of EPH1 gene of Ipomoea nil to synthesize dsRNA for RNAi. To product dsRNA, this sequence is inserted in L4440 plasmid, and transformed into HT115(DE3).

purpose

This is a gene for a transcription factor that positively regulates PCD in Ipomoea nil. Silencing this gene is expected to extend the flowering time of Ipomoea nil.

Usage and Biology

RNAi
RNAi (RNA interference) is a process in which externally introduced dsRNA suppresses the expression of genes that have complementary sequences to the dsRNA.

L4440
L4440 is a plasmid vector having two convergent T7 promoters adjacent to the multi-cloning site. By inserting a portion of the target gene sequence into the multi-cloning site of this plasmid, the target sequence is transcribed from both sides, and dsRNA can be obtained when both parts anneal.

HT115(DE3)
HT115 (DE3) is an RNase III-deficient E. coli strain that has been modified to express T7 RNA polymerase from an IPTG-inducible promoter. It lacks dsRNA-specific RNase III, which allows it to produce high levels of specific dsRNA. These attributes allow HT115 (DE3) to be a promising strain for the preparation of large amounts of viral dsRNA in vivo.

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 9
  
• 1000
  COMPATIBLE WITH RFC[1000]

cloning

This part was inserted in L4440. The L4440 has two t7 promoters, and this ​part are transcribed from both sides.

Reference

1 Andongma, A.A., Greig, C., Dyson, P.J., Flynn, N., and Whitten, M.M.A. (2020). Optimization of dietary RNA interference delivery to western flower thrips Frankliniella occidentalis and onion thrips Thrips tabaci. Arch. Insect Biochem. Physiol. 103, e21645.