Part:BBa_K2912017

Refractile inclusion bodies_ Caedibacter

Refractile inclusion bodies SZU-China 2019 iGEM team was going to find a suicide switch inside the E coli that can break the whole body of the bacteria leading to the release of RNAi molecules transcribed from E coli inducing by IPTG or some other else. Therefore, we were in need the useful mechanism. Fortunately, we finally found the Refractile inclusion bodies (R-bodies) to kill the E coli, causing the inclusion to flow out of the plasma membrane, so that we can get the RNAi molecules transcribed by E coli. Refractile inclusion bodies, known as R bodies, are produced by only a few species of bacteria. These inclusion bodies are highly insoluble protein ribbons, typically seen coiled into cylindrical structures within the cell[1]. R-bodies are produced by Paramecium endosymbionts belonging to the genus Caedibacter. These intracellular bacteria confer upon their hosts a phenomenon called the killer trait[2]. This is one of the DNA sequences for the R body locus (reb) from Caedibacter taeniospiralis. The R bodies of C. taeniospiralis are type 51. They are about 0.5 μm wide, have a maximum length of 20 μm, and 13 nm thick, possess acute angles at each end, and unroll in a telescopic fashion when exposed to a pH of 6.5 or lower. These proteinaceous ribbons are rolling up inside the cell to form a hollow cylinder about 0.5 μm in diameter and 0.5 μm long. Sequence and Features

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 15
  Illegal NheI site found at 38
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

SZU-China 2019 iGEM team

SZU-China 2019 iGEM team was going to find a suicide switch inside the E. coli that can break the whole body of the bacteria leading to the release of RNAi molecules transcribed from E. coli inducing by IPTG or some other else. Therefore, we required a useful mechanism. Fortunately, we finally found the Refractile inclusion bodies (R-bodies)to kill the E. coli, causing the inclusion to flow out of the plasma membrane so that we can get the RNAi molecules transcribed by E. coli (Fig.1).

Fig.1 Synthesis of our RNAi molecules

Refractile inclusion bodies

Refractile inclusion bodies, known as R bodies, are produced by only a few species of bacteria. These inclusion bodies are highly insoluble protein ribbons, typically seen coiled into cylindrical structures within the cell[1]. R-bodies are produced by Paramecium endosymbionts belonging to the genus Caedibacter. These intracellular bacteria confer upon their hosts, a phenomenon called the killer trait[2]. The R bodies of C. taeniospiralis are type 51. They are about 0.5 μm wide, have a maximum length of 20 μm, and 13 nm thick, possess acute angles at each end and unroll in a telescopic fashion when exposed to a pH of 6.5 or lower. These proteinaceous ribbons are rolling up inside the cell to form a hollow cylinder about 0.5 μm in diameter and 0.5 μm long[3].

Usage and Characterization

SZU-China 2019 iGEM team constructed a plasmid with BBa_K1475900 (Promoter),BBa_K2912014 (Attenuator),BBa_K2912000 (Reb A),BBa_K2912001 (Reb B),BBa_K2912003 (Reb D),BBa_K2912002 (Reb C),BBa_B0010 (Terminator)(Fig.2).

Fig.2 Plasmid construction of R-body

The attenuator was designed to control the expression of R-body via the concentration of Tryptophan. When the concentration of Trp is under 0.3%, R-body begins to express. Hence, after our RNAi molecules are adequately transcribed inside E. coli, the R-body proteins can be induced to translate to form the R-bodies rolled-up inside the bacteria. Then, we will change the pH of the media to induce the R-bodies to unroll and crack the E. coli after the R-bodies are fully expressed.

Fig.3 The Regulation of Trp Attenuator

Fig.4 The Regulation of pH on R-Bodies

The R-bodies proteins were predicted to fully translate after 44 minutes. Click SZU-China 2019 Model_R-bodyto see more.

SZU-China 2019 iGEM team has synthesized the R-bodies and taken the scanning electron microscope pictures of the R-bodies under pH=7 and pH=6 (Fig.5,6).

Fig.5 SEM pictures of the R-bodies under pH=7

Fig.6 SEM pictures of the R-bodies under pH=6

References

[1] Pond FR, Gibson I, Lalucat J, et al. R-body-producing Bacteria.[J]. Microbiol Rev, 1989, 53(1): 25-67.

[2] Matsuoka JI, Ishizuna F, Kurumisawa K, et al. Stringent Expression Control of Pathogenic R-body Production in Legume Symbiontazorhizobium Caulinodans[J]. Mbio, 2017, 8(4): 0-17.

[3] Heruth DP, Pond FR, Dilts JA, et al. Characterization of Genetic Determinants for R Body Synthesis and Assembly in Caedibacter Taeniospiralis 47 and 116.[J]. Journal of Bacteriology, 1994, 176(12): 3559-3567.