Part:BBa_K3815011

Part of the V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi
This is a section of V-ATPaseB gene of Frankliniella occidentalis to synthesize dsRNA for RNAi. To product dsRNA this sequence inserted in L4440 plasmid, and transformed into HT115(DE3).

purpose

We focused on feeding RNAi to kill these insects, based on previous studies showing that oral ingestion of dsRNA can induce RNAi-induced gene knockdown in Frankliniella occidentalis[1].
Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 452
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 320
  

cloning

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

production and purification methods

Fig1.
(1 VATPase 2 VATPase 3 EPH1 4 L4440(vector) 5 VATPase1 6 VATPase2 7 EPH1 8 L4440(vector)
RNA was produced in 1L LB medium and eluted in 100ul of water. )

We product ds RNA and purify this [2].
The 1L LB culture was started at 37C and IPTG (final 0.2mM) was added when OD exceeded 0.4 to induce RNA production for 4 hours.
E. coli was disrupted and precipitated with NaCl, and RNA contained in the supernatant was collected by ethanol precipitation. After that, phenol-chloroform treatment was performed to purify RNA.

Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.

We could confirm RNA production.