Part:BBa_K3773526:Design

Circuit to report translation in vitro

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 102
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 155
  

Design Notes

We use the constitutive p70a promoter and associated RBS because of its usefulness in cell-free systems¹. We also use this sfGFP sequence for its previous success in cell-free systems², and this terminator for the same reason¹.

The sequence inputted as a scar after the RBS is a spacer which has been shown to allow for expression with BBa_B0034 and sfGFP³.

UNS 1 and UNS 10 flank this part in order to allow for easy Gibson assembly as detailed by Torella et al., 2014⁴

Source

Marshall, R., & Noireaux, V. (2019). Quantitative modeling of transcription and translation of an all-E. coli cell-free system. Scientific reports, 9(1), 1-12.

References

¹Marshall, R., & Noireaux, V. (2019). Quantitative modeling of transcription and translation of an all-E. coli cell-free system. Scientific reports, 9(1), 1-12.

²Gräwe, A., Dreyer, A., Vornholt, T., Barteczko, U., Buchholz, L., Drews, G., ... & Kalinowski, J. (2019). A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs. PloS one, 14(3), e0210940.

³Clifton, K. P., Jones, E. M., Paudel, S., Marken, J. P., Monette, C. E., Halleran, A. D., ... & Saha, M. S. (2018). The genetic insulator RiboJ increases expression of insulated genes. Journal of biological engineering, 12(1), 1-6.

⁴Torella, 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.