Part:BBa_K4226000

3WJ-Bro

3WJ-Bro is used as a protein- independent reporter system based on a fluorescent RNA aptamer. It is applied as a gene marker in creating a system for reporting the presence and expression of target genes. The 3WJ-Bro can be used to ligate fluorescent aptamers to examine the RNA synthesis of target genes at transcriptional level in Escherichia coli cells, obviating the need for accumulation of foreign proteins.

Usage and Biology

Characterized by CAFA_China 2022

• We designed the genetic circuit that contains sulAp (sulA promoter), Amp30E, relE gene (RelE toxin), mScarlet-I (mSarlet-I) and 3WJ-Bro, and transferred it into DH10B competent cells (The control group did not contain the Amp30E Amplification Device). The gene circuit diagram is shown bellow:

pSB1C3-sulAp-Amp30E-relE-mScarlet-I-3WJ-Bro

• After bacterial culture and UV induction, we measured the OD600 and fluorescence values under 485/510nm. Then, the fluorescence per OD was calculated.
  
• The curve of experimental group was significantly higher than that of control group and the amount of relE RNA synthesis gradually increased with incubation time. According to the results, the 3WJ-Bro can be used to ligate fluorescent aptamers to examine the relE RNA synthesis at transcriptional level.

Figure: 3WJ-Bro was ligated with fluorescent aptamers in order to examine the level of relE RNA synthesis (485/510nm). Control group: pSB1C3-sulAp-relE-mSarlet-l-3WJxBro; experimental group: pSB1C3-sulAp-Amp30E-relE-mSarlet-l-3WJxBro.

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 AgeI site found at 667
  
• 1000
  COMPATIBLE WITH RFC[1000]

Reference
[1] Bai, Jiuyuan, et al. "A protein-independent fluorescent RNA aptamer reporter system for plant genetic engineering." Nature communications 11.1 (2020): 1-14.