Coding

Part:BBa_K4275028

Designed by: Haoming Qin   Group: iGEM22_GreatBay_SCIE   (2022-10-09)


eforRED-dockerin I

This composite part is consisted of a type i dockerin and a eforRED chromoprotein fused at the N terminal of the dockerin. The dockerin is a highly symmetrical and non-catalytic domain and it specifically binds with type i cohesin on the scaffoldin by high-affinity and non-covalent interactions. In cellulosome, the type one dockerins are usually fused on enzymes thus enabling the assembly of various cellulase on the CipA scaffoldins to ensure enzymatic synergies. The eforRED protein (originates from Echinopora forskaliana ) [2] fused at the N terminal of the dockerin is a reporter molecule, serving the purpose of testing whether the cohesin and the dockerin are successfully bind. By centrifugation, the successfully combined complex will display a red fluorescent.

GreatBay SCIE--3D eforRED-Doc1.png

Figure 1 The 3D structure of the protein predicted by Alphafold2.

Usage and Biology

Type i dockerin is a module which anchors the catalytic subunits to the scaffoldins. It is originally found in anaerobic bacteria C.thermocellum[1]. Its sequence is a tandem duplication of a 22-residue segment and it displays internal two-fold symmetry, consisting of a duplicated F-hand motif (a calcium-binding loop preceding an alpha helix)[1]. As a result, it requires calcium ions to be presented due to the the calcium-binding motif of the dockerin domain whose sequence resembles the EF-hand motif of calcium-binding proteins. The binding of type i dockerin and cohesin also highly relies on the polar interactions between Ser-45 and Thr-46[1].


eforRED as A Reporter Protein

The nanobody-antigen interaction was verified by mixing intact E.coli cells displaying Neae-Nb3 with the supernatant of Ag3-eforRED (Fig. 2A). Red fluorescent characteristics were observed in the pellets after resuspending the centrifuged mixture, which is absent in the control group that only contains Neae-Nb3 (Fig. 3C).

After that, the type II cohesin-dockerin interaction was tested using the mixture of Neae-Nb3, OlpB-Ag3, and the type II dockerin fused with eforRED (Fig. 2B). A negative control lacking OlpB-Ag3 was set up for result comparison. Centrifugation was used to remove supernatant and the red fluorescence was only identified in pellets of the sample group, confirming the type II cohesin-dockerin interaction (Fig. 3D).

Finally, the association between type I cohesin and type I dockerin was validated using the mixture of Neae-Nb3, OlpB-Ag3, CipA1B2C, and DocI-eforRED (Fig. 2C), red fluorescence was detected in the resuspended mixture while it was not observed in the control group lacking the primary scaffold CipA1B2C (Fig. 3E), verifying the type I cohesin-dockerin interaction.


Figure 2: Cellulosomal scaffold system construct (A) Antigen-nanobody interaction between Nb3 and Ag3 domain reported by the ligated eforRed fluorescent domain (B) Type II cohesin -dockerin interaction between a fixed secondary scaffoldin component and a type II dockerin domain reported by the ligated eforRed fluorescent domain (C) Type I cohesin-dockerin interaction between a fixed primary scaffoldin component and a type I dockerin domain reported by a ligated eforRed fluorescent domain (D) Genetic circuit designed for the expression of ligated form of Ag3, type I dockerin and type II dockerin domains fused with an eforRed fluorescent domain at N terminus for reporting the adhesive functions of those domains.



Figure 3: Production and assay of the scaffold proteins (A) SDS-PAGE analysis for the presence of type I dockerin-eforRed (B) SDS-PAGE analysis of Ag3-eforRed and type II dockerin -eforRed containing type II dockerin fused with an eforRed domain (C) The fluorescence indication for antigen-nanobody interaction for E.coli surface display with an Ag3 control group and a sample group (D) The fluorescence indication for type II cohesin-dockerin interactions with a type II dockerin control group and a sample group (E) The fluorescence indication for type I cohesin-dockerin interactions with a type I dockerin control group and a sample group.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Brás, Joana L.A., et al. “Escherichia Coli Expression, Purification, Crystallization, and Structure Determination of Bacterial Cohesin–Dockerin Complexes.” Cellulases, 2012, pp. 395–415, 10.1016/b978-0-12-415931-0.00021-5.

2. Part: "Bba K592012 - Parts.Igem.Org". Parts.Igem.Org, 2022, https://parts.igem.org/Part:BBa_K592012.


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