Difference between revisions of "Part:BBa K2912001"

Revision as of 13:41, 11 October 2022

The major structural subunit of R bodies-RebB

2019 SZZU-China

Biology

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 of 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.

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. 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. This is one of the DNA sequences for the R body locus (reb) from Caedibacter taeniospiralis.

Reb B appears to be the major structural subunit of R bodies. There're 12 species of RebB can be identified and Reb B has two molecular weights, each with six isoelectric points. Compared to Reb A and Reb C, The DNA sequence-based prediction that RebB is neutral was confirmed by observed migration of the polypeptides in isoelectric focusing gels. Although All three(Reb A, Reb B, Reb C) proteins are required for the major polymerization event, only RebB and RebC are necessary for the basic polymerization process.

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[4]. For more information, please see BBa_K2912017-R-body.

Sequence

Sequence and Features

2022 SZU-China

The 2022 SZU-China team conducted more in-depth research on the nature of the protein. We used a variety of bioinformatics tools to help us understand this protein. We hope that this survey will provide a wealth of information for other teams who need to use R-body to help them use it.

Basic physical and chemical properties

Number of amino acids: 105

Molecular weight: 10965.21

Theoretical pI: 6.52

Total number of negatively charged residues (Asp + Glu): 5 Total number of positively charged residues (Arg + Lys): 5

Extinction coefficients:

This protein does not contain any Trp residues. Experience shows that this could result in more than 10% error in the computed extinction coefficient.

Extinction coefficients are in units of M(-1) cm(-1), at 280 nm measured in water.

Ext. coefficient 1490 Abs 0.1% (=1 g/l) 0.136

Instability index:

The instability index (II) is computed to be 28.93 This classifies the protein as stable.

Aliphatic index: 77.24

Grand average of hydropathicity (GRAVY): -0.222

Domain

3D structure

According to AlphaFold structure predictions, the protein structure below has been predicted by DeepMind with AlphaFold (Jumper, J et al. 2021). For more information and additional features, please visit this sequence's page at AlphaFold DB.

Taxonomy

Interaction

number of nodes: 11

number of edges: 40

average node degree: 7.27

avg. local clustering coefficient: 0.89

expected number of edges: 10

PPI enrichment p-value: 2.46e-12

Reference

[1]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.

[1]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]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.

[4]Pond FR, Gibson I, Lalucat J, Quackenbush RL. R-body-producing bacteria.Microbiol Rev. 1989 Mar;53(1):25-67. doi: 10.1128/mr.53.1.25-67.1989. PMID:2651865; PMCID: PMC372716.

[5] Winter MA, Guhr KN, Berg GM. Impact of various body weights and serumcreatinine concentrations on the bias and accuracy of the Cockcroft-Gaultequation. Pharmacotherapy. 2012 Jul;32(7):604-12. doi:10.1002/j.1875-9114.2012.01098.x. Epub 2012 May 10. PMID: 22576791.

[6]Matsuoka JI, Ishizuna F, Kurumisawa K, Morohashi K, Ogawa T, Hidaka M, SaitoK, Ezawa T, Aono T. Stringent Expression Control of Pathogenic R-body Productionin Legume Symbiont Azorhizobium caulinodans. mBio. 2017 Jul25;8(4):e0071517. doi: 10.1128/mBio.00715-17. PMID: 28743814; PMCID:PMC5527310.

[7]White DW, Tartaglia LA. Leptin and OB-R: body weight regulation by a cytokinereceptor. Cytokine Growth Factor Rev. 1996 Dec;7(4):303-9. doi:10.1016/s1359-6101(96)00040-8. PMID: 9023054.

[8]Matsuoka JI, Ishizuna F, Ogawa T, Hidaka M, Siarot L, Aono T. Localization ofthe reb operon expression is inconsistent with that of the R-body production inthe stem nodules formed by Azorhizobium caulinodans mutants having a deletion of praR. J Gen Appl Microbiol. 2019 Sep 14;65(4):209-213. doi:10.2323/jgam.2018.09.003. Epub 2019 Feb 5. PMID: 30726794.