Part:BBa_K4765108

Twister P1 + T7_RBS + INPNC-Nb3 fusion + stem-loop

contributed by Fudan iGEM 2023

INPNC-Ag3 fusion is composed of a surface display system (INPNC+linker) and the coding sequence of a nanobody. INPNC exhibits compatibility with the translocation and surface display of proteins containing multiple cofactors and disulfide bond-containing passengers^[1].Ag3 is a corresponding antigen of BBa_K4765007(Nb3)^[2]. The interaction between Ag-Nb can mediate specific adhesion of Escherichia coli. A flexible protein domain linker of 10 aa was introduced between INPNC and Ag3 to ensure independent functionality of Ag3 and INPNC with minimal mutual disruption.

Usage and Biology

The surface-displayed antigen can specifically interact with the nanobody produced by BBa_K4765107.

Charaterization

Sequencing map

Figure 1. Sequencing map of INPNC-Nb3 fusion

Sequencing is performed using the primer：Kan-F: 5-ATTCTCACCGGATTCAGT-3.

Successful Protein Expression

Figure 2. SDS-PAGE electrophoresis of INPNC-Ag3 and INPNC-Nb3

We constructed INPNC-Ag3 and INPNC-Nb3 into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lane 1 to 2 represent INPNC-Ag3 and INPNC-Ag3 + IPTG, Lane 3 to 4 represent INPNC-Nb3 and INPNC-Nb3 + IPTG. As indicated by the red arrow, we successfully expressed INPNC-Ag3 and INPNC-Nb3.

Selection through Aggregation Assay

To identify a suitable protein for surface display, we conducted a comparative analysis of the surface display efficiency of intimin and INPNC by assessing their biofilm formation capabilities using an aggregation experiment.

Specifically, bacterial solutions of intimin-Ag3 and intimin-Nb3, INPNC-Nb3 and intimin-Ag3, INPNC-Ag3 and intimin-Nb3, as well as plain E.coli (control), were mixed in a 1:1 ratio (600μL per strain per tube, with 1.2mL for plain bacteria aggregation) and allowed to settle. Sampling was performed at 0, 3, and 6 hours by collecting 100μL aliquots from the upper 25% of each mixture (supernatant) in each tube. These samples were subsequently transferred to EP tubes and stored at 4℃ until the final sampling. Afterward, they were resuspended and transferred to a 96-well assay plate for OD~600~ measurement. The percentage of bacteria remaining in the supernatant at 3 hours was determined by dividing the bacterial count at 3 hours (as determined by the OD~600~ measurement) by the bacterial count at 0 hours.

As shown in Figure 2 and 3, when both the antigen and nanobody select intimin as the display protein, the results demonstrate significantly better performance compared to other combinations. This suggests that the surface display efficiency of intimin surpasses that of INPNC.

Figure 2. Aggregation Experiment Results

From left to right: intimin-Ag3/Nb3, INPNC-Ag3/intimin-Nb3, and intimin-Ag3/INPNC-Nb3 results three hours after mixing.

Figure 3. Bacteria Percentage Remaining in the Supernatant at 3 Hours

The bacterial quantity in the supernatant is reflected by measuring the OD~600~ (1 OD~600~ corresponds to 10^8 bacterial particles).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NotI site found at 542
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 391
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 133
Illegal NgoMIV site found at 466
Illegal AgeI site found at 490
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1199

Reference

↑ van Bloois, E., Winter, R. T., Kolmar, H., & Fraaije, M. W. (2011). Decorating microbes: Surface display of proteins on Escherichia coli. Trends in Biotechnology, 29(2), 79–86. https://doi.org/10.1016/j.tibtech.2010.11.003
↑ Glass, D. S., & Riedel-Kruse, I. H. (2018). A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell, 174(3), 649-658.e16. https://doi.org/10.1016/j.cell.2018.06.041

[1] van Bloois, E., Winter, R. T., Kolmar, H., & Fraaije, M. W. (2011). Decorating microbes: Surface display of proteins on Escherichia coli. Trends in Biotechnology, 29(2), 79–86. https://doi.org/10.1016/j.tibtech.2010.11.003

[2] Glass, D. S., & Riedel-Kruse, I. H. (2018). A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell, 174(3), 649-658.e16. https://doi.org/10.1016/j.cell.2018.06.041

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