Difference between revisions of "Part:BBa K4593021:Design"

Latest revision as of 00:03, 9 October 2023

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

@@ Line 10: / Line 10: @@
 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.