Part:BBa_K5121011

Reb1 Locus

Biology

Refractile bodies, known as R bodies, are ribbon-like protein complexes produced by certain strains of bacteria. Five classes of R bodies have been described — this part specifically encodes a modified type 51 R body containing four genes; rebA, rebB, rebC, and rebD. rebA and rebB constitute the primary structural components of R bodies, while rebC is thought to aid in the polymerisation process — the function of rebD remains unknown (Heruth et al., 1994). Under basic conditions, R bodies exist in a coiled-up conformation, but will extend in a telescopic fashion under acidic conditions (Heruth et al., 1994). In nature, R bodies are produced by bacterial endosymbionts of some Paramecia. Also referred to as kappa particles, these bacteria constitute the genus Caedibacter (Beier et al., 2002). These bacterial endosymbionts confer a killer trait to host paramecia — when released and taken up by sensitive paramecia, the bacteria are exposed to an acidifying environment in the endosome (Figure 0.1). These conditions cause the extension of R bodies inside the bacteria, rupturing them and the endosome to release a toxin to kill the host cell (Pond et al., 1989). Their ability to burst endosomes make R bodies appealing candidates for use in drug delivery, as they could hold the key to solving the endosomal escape problem.

Figure 0.1. R body mechanism in nature as illustrated by Polka and colleagues (2016). The unique ability of R bodies to burst endosomes makes them attractive candidates for nano-based drug delivery.

Design notes

Although in vivo, the reb genes are transcriptionally independent, here they are encoded in a polycistronic fashion.

The reb locus was integrated into a Reb1 plasmid (Figure 1) with a T7 promoter and terminator, containing a lacI gene, as well as a pBR322 and f1 origin. Transformed bacteria were selected for using a kanamycin resistance (kanR) gene. DH5α cells were used for cloning, while BL21 cells were used for expressing R bodies.

Figure 1. Plasmid map of the Reb1 plasmid used to express Type 51 R bodies.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1481
  Illegal NheI site found at 1543
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1423
  Illegal XhoI site found at 1721
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

Expression of this part was reliable and consistently produced large quantities of R bodies. Expression of functional R bodies was less reliable, as there appeared to be a large quantity of R bodies that did not extend upon addition of acid. We found that R bodies were quite prone to clumping, particularly in the extended state, perhaps due to an increase in exposed surface area (Figure 2). We therefore recommend vortexing samples prior to imaging.

Figure 2. Clumping pattern of R bodies over time.

The purpose of our project is to engineer these proteins for drug delivery, harnessing their pH-sensitive extension properties to facilitate endosomal escape. In lieu of this aim, we made several modifications to the rebA and rebB domains that each facilitate a unique conjugation strategy. We also sought to conjugate dyes to unmodified R-bodies. We utilised click-chemistry to achieve this aim.

Treating R-bodies with 2-pyridinecarboxaldehyde (2PCA) selectively target the N-terminus, generating an alkyne group. This alkyne group is then compatible with CuAAC of an azide containing dye, sulfo-Cy5. This strategy proved successful (Figure 3).

Figure 3. 2-PCA approach for conjugation.A) 5-ethynylpicolinaldehyde conjugation and the CuAAC click reaction of sulfo-Cy5 onto alkyne-functionalised R bodies is shown. B) Control pellet (left) compared to experimental pellet (right) after three 20% ethanol washes. The control pellet is white whereas the experimental pellet is blue, indicating successful conjugation.

References

Beier, C. L., Horn, M., Michel, R., Schweikert, M., Görtz, H.-D., & Wagner, M. (2002). The Genus Caedibacter Comprises Endosymbionts of Paramecium spp. Related to the Rickettsiales (Alphaproteobacteria) and to Francisella tularensis (Gammaproteobacteria). Applied and Environmental Microbiology, 68(12), 6043–6050.

Chen, I., Dorr, B. M., & Liu, D. R. (2011). A general strategy for the evolution of bond-forming enzymes using yeast display. Proceedings of the National Academy of Sciences - PNAS, 108(28), 11399–11404. Heruth, D. P., Pond, F. R., Dilts, J. A., & Quackenbush, R. L. (1994). Characterization of genetic determinants for R body synthesis and assembly in Caedibacter taeniospiralis 47 and 116. Journal of Bacteriology, 176(12), 3559–3567.

Polka, J. K., & Silver, P. A. (2016). A Tunable Protein Piston That Breaks Membranes to Release Encapsulated Cargo. ACS Synthetic Biology, 5(4), 303–311.

Pond, F. R., Gibson, I., Lalucat, J., & Quackenbush, R. L. (1989). R-body-producing bacteria. Microbiological Reviews, 53(1), 25–67.