Part:BBa_K3773512:Design

Circuit to report transcription in vivo (alternate design)

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]

Design Notes

After a thorough literature search, it became clear that the best method with which to sense transcriptional changes was through aptamers. When deciding which aptamer to use for our sensor, we focused on three aptamers specifically: Mango, Spinach, and Broccoli. These aptamers are widely regarded as “the most promising fluorescent RNA aptamers”¹. As Broccoli binds to a non-toxic, membrane-permeable dye (DFHBI-1T) that has minimal effects on host activities, is widely used in vivo, and has a relatively simple testing procedure, our team decided to use Broccoli as our aptamer². However, we still needed to choose which form of Broccoli to use. We eventually decided on F30-Broccoli, as it has been shown that its scaffold is not recognized by nucleases for degradation like other aptamers, that it is regarded as the “best-performing aptamer,” and that it is widely used^{3, 4}. Due to all these factors, our team decided to use F30-Broccoli. Using this aptamer, we designed two transcriptional burden sensors. Our first design places control of F30-Broccoli expression under the strong, constitutive Anderson promoter BBa_J23119, without incorporation of any RBS or spacers. Our second design is this part, BBa_ K3773512. It places control of F30-Broccoli expression under the strong, constitutive P70a promoter without incorporation of any RBS or spacers. This is because P70 has been successfully used in vivo to drive transcription of a split Broccoli aptamer⁵. In both circuits, F30-Broccoli is directly followed by the strong, synthetic, bidirectional terminator BBa_B1006.

UNS 1 and UNS 10 flank this part in order to allow for easy Gibson assembly as detailed by Torella et al., 2014⁶

Source

See basic parts.

References

¹Shanaa, O., Rumyantsev, A., Sambuk, E., & Padkina, M. (2021). In Vivo Production of RNA Aptamers and Nanoparticles: Problems and Prospects. Molecules. 26(5): 1422,  https://doi.org/10.3390/molecules26051422

²Okuda, M., Fourmy, D., & Yoshizawa, S. (2017). Use of Baby Spinach and Broccoli for imaging of structured cellular RNAs. Nucleic Acids Research, 45(3):1404-1415. https://doi.org/10.1093/nar/gkw794

³Filonov, G., Kam, C., Song, W., and Jaffery S. (2015).  In-gel imaging of RNA processing using broccoli reveals optimal aptamer expression strategies. Chem Biol. 22(5). Doi: 10.1016/j.chembiol.2015.04.018.

⁴Thorn, K. (2017). Genetically encoded fluorescent tags. Molecular Biology of the Cell. 28(7). https://doi.org/10.1091/mbc.e16-07-0504

⁵Alam, K. K., Tawiah, K. D., Lichte, M. F., Porciani, D., & Burke, D. H. (2017). A fluorescent split aptamer for visualizing RNA–RNA assembly in vivo. ACS synthetic biology, 6(9), 1710-1721.

⁶Torella, J. P., Boehm, C. R., Lienert, F., Chen, J. H., Way, J. C., & Silver, P. A. (2014). Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly. Nucleic acids research, 42(1), 681-689.