Part:BBa_K5121024

Reb1: RebB C-terminal Cys

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 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).

Figure 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.

Part overview

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. This composite part encodes an R body compatible with cysteine maleimide conjugation at the C-terminus of rebB — facilitating the attachment of hundreds of thousands of drug molecules down the length of assembled R bodies. See the design page for more information.

Cysteine maleimide conjugation is a form of Michael addition, in which the thiol of the cysteine acts as a nucleophile to react with maleimide, forming a thiosuccinimide adduct (Figure 2). Through this reaction, drugs with maleimide groups can hence be reacted onto proteins with readily accessible cysteines.

Figure 2. Cysteine maleimide conjugation reaction mechanism. This conjugation reaction can be used to attach hundreds of thousands of drug molecules down the length of assembled R bodies.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 806
  Illegal NheI site found at 868
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 748
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterisation

As described in the design section, a two-fragment PCR was run on the purified Reb1 plasmid to add the C-terminal SGGGGC motif to rebB. Two separate PCR reactions were performed with extension times dependent on the length of each fragment. The products of each PCR reaction were run on an agarose gel to determine if the PCR was successful (Figure 3A,B). After verifying the PCR, the remaining products were run on a new gel, from which the products were purified. A Gibson assembly was prepared with the purified PCR products. The Gibson assembly products were immediately transformed into DH5-alpha E. coli cells for propagation and BL21 E. coli cells for expression. Successful Gibson assembly was indicated by the growth of colonies on plates with kanamycin (Figure 3C). Plasmids from selected colonies were extracted and sequenced to verify they contained the correct modified rebB sequence.

Figure 3 Cloning of RebB C-terminal Cysteines. PCR reactions were performed in duplicate or triplicate. A) Short fragment PCR products amplifying the insert. B) Long fragment PCR products amplifying the backbone. C) DH5-Alpha cells transformed with the products of Gibson assembly of the two amplified fragments. Several colonies (circled) grew on plates containing kanamycin. These colonies should contain plasmids with our modified rebB constructs.

Successful expression and purification of R bodies was first confirmed by imaging the R bodies in resuspension buffer (Figure 4A). The pH of the resuspension buffer was 7.5, in which we expect the R-bodies to remain in a coiled conformation like their wild-type counterpart (BBa_K5121011). This was observed in our microscopy images of these samples. We next tested the extension behaviour of these modified R-bodies in pH 5 HCl (Figure 4B). These R-bodies readily extend in these conditions. Further testing of their ability to retract in basic conditions could be performed in future.

Figure 4. Purification and extension testing of RebB C-term Cys R bodies. A) Purified RebB C-term Cys R bodies R bodies in resuspension buffer (25 mM Tris pH 7.5, 100 mM NaCl) observed at 63x under phase contrast. B) RebB C-term Cys R bodies extended in pH = 5 HCl observed at 63x under phase contrast.

After confirming that the modified constructs retain their extension ability in acidic conditions, we next attempted to perform a cysteine maleimide conjugation reaction with the dye sulfo-Cy5 maleimide. Unfortunately, this construct was not successfully conjugated with the dye (Figure 5A). However, another cysteine-maleimide R-body construct engineered by our team (BBa_K5121023) was successfully conjugated in the same conditions (Figure 5B).

Figure 5. Thiol-maleimide conjugation of sulfo-Cy5 maleimide. A) Cys-C RebB (left) appears white after three MQW washes, indicating an unsuccessful conjugation. Our other cysteine-maleimide compatible construct (Cys_N RebB, right) appears as a bright blue pellet, indicating a successful conjugation. B) Cys-N RebB (left) compared to a wild-type R-body control (right) after conjugation. Both samples have been washed thrice with MQW.

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.