Difference between revisions of "Part:BBa K4275026"

Latest revision as of 11:10, 12 October 2023

Improved by Fudan iGEM 2023

To form a programmable self-assembling biofilm, different strengths of Ag/Nb pairs are required. In our project, we employed the surface display system neae(intimin) to present three pairs of Ag/Nb on the E. coli 's cell membrane and assessed their strengths. Simultaneously, we connected lectins LCA and MVN to the C-terminus of intimin, facilitating the binding between E. coli and cyanobacteria. As a result, we established a stable symbiotic platform between cyanobacteria and E. coli.

Improved part

Our improved part is BBa_K4765106 (Twister P1 + T7_RBS + intimin-Nb3 fusion + stem-loop),BBa_K4765109 (Twister P1 + T7_RBS + intimin-MVN fusion + stem-loop) and BBa_K4765110 (Twister P1 + T7_RBS + intimin-LCA fusion + stem-loop). We not only introduce three independent antigen-nanobody pairs and two lectins into the synthetic adhesion system but as well construct these parts into our ribozyme-assisted polycistronic co-expression system.

Neae-Nb3

Neae-Nb is a type of nanobody, which contains variable domains of camelid heavy-chain antibodies, that can be expressed on bacterial surfaces due to their small size (~125 amino acids) and stability under a variety of conditions[1]. The combination between the single-domain structure and the intimin autotransporter allows the entirety of a highly specific, cell surface-bound adhesin to be encoded as a single fusion protein. Neae-Nb will specifically adhere to a corresponding antigen via the Nb-Ag interaction, which can form the adhesins and control morphology and patterning of multicellular assemblies.

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

Usage and Biology

Nanobodies are the recombinant variable domains of heavy-chain-only antibodies, with many unique properties such as small size, excellent solubility and superior stability. This nanobody domain interacts with specific antigen through antigen nanobody interactions and can be used for cell-cell adhesion and bacterial surface display with a high adhesion stability.The CDR(Complementarity-determining region) domain of the nanobodies determine their antigen-recognition specificity.In our experiment, Nb3[1] displayed on the outer membrane specifically interacts with Ag3 on the OlpB-Ag3 fusion construct (BBa_K4275013), promoting the immobilization of cellulosome complexes onto the surface of the bacteria.

Characterization

Nanobody-antigen interaction

An E.coli expression vector of surface display system Neae-Nb was constructed and transformed into E.coli host cells. The export tag fused with the coding sequence directs the nanobody domain to be exported and presented on the extracellular surface of the bacteria. Interaction between the exposed nanobody domain and the antigen3 domain fused with an eforRed reporter protein was shown by the red fluorescent characteristics in the pellet formed after centrifugation, which is absent in the control group with no Nb production, meaning that it is unable to generate cohesion with ligated antigen [1]. This highly efficient cell surface adhesion approach was proven useful in a variety of fields and aid our needs for constructing complex nanomachines on the surface of E.coli.

Figure 2: Fig.2 The surface display system characterized by nanobody-antigen interaction between Neae-Nb3 and Ag3 fused with reporter protein eforRED.

Surface display system

In order to display the cellulosome complex on the surface of E.coli, we decided to use nanobody (Nb)-antigen (Ag) interaction between Neae-Nb3 and Ag3.

In the construction of Neae-Nb3 vector we fused ribozyme RiboJ, which is a genetic insulator that increases the protein expression of downstream sequence. The effect of RiboJ fusion was first verified using the construction of prha-riboJ-RFP plasmid (Fig.3A). Comparison of fluorescence intensity between prha-riboJ-RFP and control group absence of riboJ suggests significant increase in level of RFP expression (Fig.3C and Fig.3D).

The prha-riboJ-Neae-Nb3 plasmid were then constructed (Fig.3A) and transformed into E.coli BL21 strain for rhamnose-inducible expression. SDS-page was performed and Neae-Nb3 was presented in sediment, indicating the successful expression of Nb3 on bacterial cell surface (Fig.3B).

Figure 3: Fig.3 E.coli surface display system. (A) Construction of prha-riboJ-Neae-Nb3 and prha-riboJ-mRFP1 vectors. (B) SDS-page analysis for Neae-Nb3 expression. (C) Comparison of RFP fluorescence intensity between prha-riboJ-mRFP1 construct and prha-mRFP1 set as control. (D) Stronger positive correlation was shown between RFP fluorescence intensity and concentration of rhamnose in prha-riboJ-mRFP1 construct.

Verification of protein-protein interaction

In order to verify the natural function of Neae-Nb-Ag3 surface display system, we constructed E.coli expression vector for Ag3 domain ligated with eforRed domain to visualize the protein-protein interaction (Fig.4B). The Ag3-eforRED construct was cultured for IPTG-inducible expression. Desired proteins were identified in whole cell and supernatant samples as shown by SDS-page analysis (Fig.4C). Intact E.coli cells expressing Neae-Nb on their surface was mixed with eforRed-Ag3 supernatant. In contrast to the control group only with Neae-Nb, red fluorescence was observed in Neae-Nb-Ag3 mixture under blue-light condition, providing evidence for Nb-Ag3 interaction (Fig.4D).

Figure 4: Neae-Nb3-Ag3 surface display system expression and verification. (A) Antigen-nanobody interaction between Nb3 and Ag3 domain reported by the ligated eforRED fluorescent domain. (B) Genetic circuit construction of Ag3-eforRED vector. (C) SDS-page analysis of Ag3 expression. (D) The fluorescence indication for antigen-nanobody interaction of E.coli surface display with an Ag3 control group and a sample group.

Cellulosome construction

We assembled the cellulose-like complex on the surface of E.coli by adding primary scaffold proteins, cellulases and cellulase boosters onto E.coli expressing secondary scaffold proteins. The mixture was centrifuged and resuspended in tris-HCl. The mixture underwent centrifugation and resuspension using tris-HCl, and cellulose was added to the mixture. After 24h, the mixture was filtered and tested for glucose by Benedict's test. From the result, we determined that the cellulosome-like complexes are able to degrade cellulose at a higher efficiency than cell-free cellulases mixture (Fig.5A and 5B). The overall success in engineering our project was verified by the successful construction of cellulosome complex and degrading cellulose to reducing sugars.

Figure 5: Fig.5 The Benedict’s quantitative and qualitative tests for reducing sugar produced by the enzymatic or cellulosomal degradation of cellulose (A) Benedict’s qualitative test result for reducing sugar production through 24h of cellulose degradation by cellulosome, cellulosome without boosters, nanobody presenting cell+free cellulases+cellulase boosters, nanobody presenting cell+cellulases and nanobody presenting cell control from left to right (B) Benedict’s quantitative test for absorbance of the samples obtained from the Benedict’s qualitative test at 635 nm wavelength.

Sequence and Features

References

1. Glass, David S, and Ingmar H Riedel-Kruse. “A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns.” Cell vol. 174,3 (2018): 649-658.e16. doi:10.1016/j.cell.2018.06.041