Part:BBa_K5090006

miRNA-326 Target Site

Description

The reverse-complement of human miRNA-326 (hsa-miR-326), serving as a target site to enable translational gene repression with Argonaute2 (BBa_K5090000).

Usage and Biology

According to Tuschl et al. (2004), Argonaute2 (or Ago2) is capable of cleaving mRNA transcripts with a site complementary to any microRNA that Ago2 loads. The function of this target site is to enable this process to occur on the gene to which it is attached.

Characterization

Wet Lab

The 2024 Stony Brook University iGEM Team is currently attempting to physically characterize the effectiveness of this target site in an E. coli-based cell free system (S30 extract). We hope to demonstrate that when this target site, microRNA-326, and Argonaute2 are all present, that the gene which the target site is meant to assist in the repression of will indeed be repressed.

Dry Lab

In Dry Lab, we were able to characterize that in the presence of microRNA-326, Ago2 should be able to reactivate GFP expression through its suppression of LacI and L7Ae, which are under regulation of a microRNA-326 target site. This should generally hold true for other linked microRNAs and target sites, so long as the sequences are complementary.

Specifically, given an in-vivo context in E. coli, expression should range from the concentration of approximately 2.5*10^-4 M without any dual regulation, nearly 1.8*10^-10 M with dual-regulation, and 2.5*10^-4 once again when Ago2 and microRNA-326 are present to facilitate repression of the dual-regulation system.

Figures 1, 2, and 3: Graphs illustrating the effect of Ago2 upon GFP expression given GFP is placed under LacI and L7Ae regulation and LacI and L7Ae are placed under regulation of Ago2 and miRNA-326.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

• Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., & Tuschl, T. (2004). Human Argonaute2 Mediates RNA Cleavage Targeted by miRNAs and siRNAs. Molecular Cell, 15(2), 185–197. https://doi.org/10.1016/j.molcel.2004.07.007