Composite

Part:BBa_K4286504:Design

Designed by: Minxi Zeng   Group: iGEM22_SZU-China   (2022-09-17)


Improved version of Refractile inclusion body gene cluster


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1207
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1042
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1024


Design Notes

We have designed a modified version of the R-body to be used as a kill switch for engineered E. coli. The R-body gene cluster was placed on the plasmid pRSFDuet1， controlled by the arabinose promoter. We finally found that the R-body ( refractive inclusion body) killed E. coli, causing the inclusion to flow out of the plasma membrane, so that we could get RNAi molecules transcribed by E. coli. In the design of our project, we engineered E. coli to produce RNAi molecules and synthesize R-bodies. In the first step of production, we induced E. coli to produce RNAi molecules by IPTG; in the second step, we used arabinose to induce E. coli to produce R-body, and then we acidify the bacterial medium to crack E. coli， leading to the release of RNAi molecular.

Source

R-body is produced by bacteria of the genus Caedibacter or Caedimonas, which are obligate endosymbionts of Paramecium.

References

[1]Heruth DP, Pond FR, Dilts JA, Quackenbush RL. Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116. J Bacteriol. 1994 Jun;176(12):3559-67. doi: 10.1128/jb.176.12.3559-3567.1994. PMID: 8206833; PMCID: PMC205544.

[2]Wang B, Lin YC, Vasquez-Rifo A, Jo J, Price-Whelan A, McDonald ST, Brown LM, Sieben C, Dietrich LEP. Pseudomonas aeruginosa PA14 produces R-bodies, extendable protein polymers with roles in host colonization and virulence. Nat Commun. 2021 Jul 29;12(1):4613. doi: 10.1038/s41467-021-24796-0. PMID: 34326342; PMCID: PMC8322103.

[3]Koehler L, Flemming FE, Schrallhammer M. Towards an ecological understanding of the killer trait - A reproducible protocol for testing its impact on freshwater ciliates. Eur J Protistol. 2019 Apr;68:108-120. doi: 10.1016/j.ejop.2019.02.002. Epub 2019 Feb 12. PMID: 30826731.