Part:BBa_K3815012

Part of the TrxZ gene of A. thaliana to synthesize dsRNA for RNAi
This is a section of the TrxZ gene of Arabidopsis thaliana to synthesize dsRNA for RNAi. To product dsRNA, this sequence is inserted in the 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 gene will whiten leafs of Arabidopsis thaliana[1].

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 are annealed.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 442
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

cloning

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

Reference

1 Chen, Z., He, J., Luo, P., Li, X., and Gao, Y. (2018). Production of functional double-stranded RNA using a prokaryotic expression system in Escherichia coli. Microbiologyopen e787.