Device

Part:BBa_K4593021:Design

Designed by: Jianfei Song   Group: iGEM23_BNDS-China   (2023-10-08)
Revision as of 00:03, 9 October 2023 by Jianfei Song (Talk | contribs) (→‎Design Notes)

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S. aureus in vivo elimination apparatus for B. subtilis


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 439
    Illegal BglII site found at 6064
    Illegal BamHI site found at 1323
    Illegal XhoI site found at 2618
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 5716
    Illegal NgoMIV site found at 6548
    Illegal NgoMIV site found at 7222
    Illegal AgeI site found at 3117
    Illegal AgeI site found at 3255
    Illegal AgeI site found at 5585
    Illegal AgeI site found at 6692
    Illegal AgeI site found at 6920
    Illegal AgeI site found at 7091
    Illegal AgeI site found at 7204
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The promoter and RBS of the circuit are optimized for protein expression in B. subtilis. However, further codon optimization might be needed to achieve the full potential for expression level.

The P2 promoter shows a high background expression in E. coli, but the detection device should function as intended in B. subtilis, as previous research showed that the same design works in closely related gram-positive bacteria (Bacillus megaterium)[5]. However, we don't have enough time to test this design.

As the host bacteria changes, Spn1s_LysRZ may not work for B. subtilis. Thus, another self-lysing enzyme should be used as a substitution to let the circuit function in B. subtilis.

For the initial version designed for the expression in E.coli, see BBa_K4593020.

Source

LysDZ25 is from bacteriophage DZ25[1], LysGH15 is from Bacteriophage GH15[2], and ClyC is a hybrid endolysin with CBD of LysPALS1 and EAD of LysSA12[3]; Spn1s_LysRZ is from bacteriophage SPN1S[4].

The QS system is from the S. aureus genome, contributed by iGEM07_Cambridge.

References

[1] Chang, Y., Li, Q., Zhang, S., Zhang, Q., Liu, Y., Qi, Q., & Lu, X. (2023). Identification and Molecular Modification of Staphylococcus aureus Bacteriophage Lysin LysDZ25. ACS Infectious Diseases, 9(3), 497–506. https://doi.org/10.1021/acsinfecdis.2c00493

[2] Gu, J., Feng, Y., Feng, X., Sun, C., Lei, L., Ding, W., Niu, F., Jiao, L., Yang, M., Li, Y., Liu, X., Song, J., Cui, Z., Dong Soo Han, Du, C., Yang, Y., Liu, Z.-J., Liu, Z.-J., & Han, W. (2014). Structural and Biochemical Characterization Reveals LysGH15 as an Unprecedented “EF-Hand-Like” Calcium-Binding Phage Lysin. 10(5), e1004109–e1004109. https://doi.org/10.1371/journal.ppat.1004109

[3] Lee, Chanyoung, et al. “Development of Advanced Chimeric Endolysin to Control Multidrug-Resistant Staphylococcus Aureus through Domain Shuffling.” ACS Infectious Diseases, vol. 7, no. 8, 28 May 2021, pp. 2081–2092. https://doi.org/10.1021/acsinfecdis.0c00812

[4] Lim, J.-S., Shin, H., Kang, D.-H., & Ryu, S. (2012). Characterization of endolysin from a Salmonella Typhimurium-infecting bacteriophage SPN1S. Research in Microbiology, 163(3), 233–241. https://doi.org/10.1016/j.resmic.2012.01.002

[5] Marchand, N., & Collins, C. H. (2013). Peptide-based communication system enables Escherichia coli to Bacillus megaterium interspecies signaling. Biotechnology and Bioengineering, 110(11), 3003–3012. https://doi.org/10.1002/bit.24975