Composite
clpB senso

Part:BBa_K3773518:Design

Designed by: Justin Berg   Group: iGEM21_William_and_Mary   (2021-10-15)


Circuit to report PclpB expression 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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 275
    Illegal AgeI site found at 596
  • 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 uses BBa_B0034 as an RBS. The original version, BBa_K3773517, utilizes the RBS-containing region which normally follows the promoter for clpB in E. coli.

We made this part because we wanted to improve upon the original version, which was only half as fluorescent as the positive control, BBa_K3773513, when we performed our initial functional confirmation of this circuit. More importantly, BBa_K3773517 did not succeed at producing higher fluorescence when cotransformed alongside another circuit. Fortunately, we had considered that BBa_K3773517 might have produced lower fluorescence than was possible, and therefore designed a different version of the clpB sensor circuit which includes the RBS BBa_B0034 rather than the RBS native to E. coli. This design, K3773518, is more in line with our BBa_BK3773513, which successfully produced fluorescence and includes BBa_B0034.

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. Therefore, we consider this part (BBa_K3773518) an improvement upon BBa_K3773517. Additionally, when we cotransformed BBa_K3773518 with pBbB8k-csg-amylase into NEB5-alpha cells, we did see that fluorescence was higher over time for cotransformed cells than cells with the circuit alone. This suggests that, unlike BBa_K3773517, BBa_K3773518 successfully responds to the presence of pBbB8k-csg-amylase with increased sfGFP expression.


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.