Part:BBa_K3815012
Part of the TrxZ gene of A. thaliana to synthesize dsRNA for RNAi
This is a section of Trx-Z gene of Arabidopsis thaliana to synthesize dsRNA for RNAi. To product dsRNA this sequence inserted in L4440 plasmid, and transformed into HT115(DE3).
purpose
Trx-Z is a gene responsible for chloroplast formation during early development in Arabidopsis thaliana. Silencing this genes is whitening leaf of Arabidopsis thaliana.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 442
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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
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 purificated by phenol-chloroform treatment.
Electrophoresis in acrylamide gel was performed to confirm whether the target RNA was produced.
We could confirm RNA production.
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