Part:BBa_K3773517:Design

Circuit to report PclpB expression in vivo

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 275
  Illegal AgeI site found at 587
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

We found that of various heat shock proteins, clpB is one of the most frequently differentially expressed (it is upregulated) in E. coli upon transformation with a heterologous circuit. As a result, we use this gene as one representative of genes related to the heat shock response.

We created two versions of this circuit. This circuit utilizes the RBS-containing region which normally follows the promoter for clpB in E. coli. However, the alternate version, BBa_K3773518, uses BBa_B0034 in its place. Upon growth of an overnight culture and measurement of fluorescence in a plate reader, we found that BBa_K3773518 has about a fourfold higher fluorescence compared to BBa_K3773517. While BBa_K3773518 fluoresced about twice as much as the positive control, BBa_K3773513, BBa_K3773517 only fluoresced about half as much.

The sequence inputted as a scar after the RBS is a spacer which has been shown to allow for expression with Bba_B0034 and sfGFP¹.

UNS 1 and UNS 10 flank this part in order to allow for easy Gibson assembly as detailed by Torella et al., 2014².

Source

See basic parts

References

¹Clifton, 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.

²Torella, 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.