Part:BBa_K4765106
Twister P1 + T7_RBS + intimin-Nb3 fusion + stem-loop
Contents
Introduction
We introduced a self-assembly synthetic adhesion system by transfecting initimin-Nb2 fusion into E. coli. Initimin-Nb3 fusion is composed of a surface display system (intimin) and the coding sequence of a nanobody. The surface display system, which includes a short N-terminal signal peptide to direct its trafficking to the periplasm, a LysM domain for peptidoglycan binding, and a beta-barrel for transmembrane insertion[1], possesses the outer membrane anchoring of the nanobody[2].
Usage and Biology
The surface-displayed nanobody can specifically interact with the antigen produced by BBa_K4765105 .In our project, we took full advantage of the Ag-Nb interaction to create a bacteria lawn with a programmable physical structure[3].
Characterization
To confirm biofilm formation through intimin-Ag/Nb, we employed both **aggregation experiments** and **fluorescence microscopy imaging** to demonstrate its ability to mediate biofilm formation.
Aggregation Asssay
In the aggregation experiment, bacterial solutions of aTc-induced/not induced intimin-Ag3 and intimin-Nb3, intimin-Ag2 and intimin-Nb2, intimin-Ag1 and intimin-Nb1 E.coli, were mixed in a 1:1 ratio (600μL per strain per tube) and allowed to settle. Sampling was performed at 0 and 3 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 is shown in Figure 2 and 3 , at 3 hours, in all the aTc-induced *E. coli* samples, bacteria percentage remaining in the supernatant was significantly lower compared to the uninduced samples. And among them, the Ag3/Nb3 pairs exhibited the most favorable performance.
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| Figure 2. 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). |
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| Figure 3. Aggregation Experiment Results at 3 Hours
From left to right: aTc-induced intimin-Ag1/Nb1, not-induced intimin-Ag1/Nb1, aTc-induced intimin-Ag2/Nb2, not-induced intimin-Ag2/Nb2 and aTc-induced intimin-Ag3/Nb3, not-induced intimin-Ag3/Nb3. |
Fluorescence Microscopy Imaging
We also employed microscopy imaging to observe the growth and expansion of biofilm. Glass slides were treated with PDL (Poly-D-Lysine) for 10 seconds, followed by mixing *E. coli* expressing intimin-Ag3 and intimin-Nb3-mScarlet on these slides. After several washes with LB KanR medium, 100 μL of LB KanR medium was added. The location of the founder cell was determined, and imaging was initiated on the microscope stage at 25°C, with video recordings captured at 5-minute intervals，and photographs taken at 0, 2, and 5.5 hours.
As illustrated in figure 4, the presence of Ag/Nb pairs on the surface enables two different strains of bacteria to coexist harmoniously by attaching to each other in an appropriate ratio. This coexistence is evident even at 5.5 hours, as both strains of bacteria remain within the field of view.
In the video that follows, we present additional evidence of bacterial growth and division within our biofilm, where bacteria bound by Ag/Nb pairs can be observed continuously dividing. The fluorescent cells in the video consistently undergo cell division throughout the entire recording.
These results collectively demonstrate that intimin-Ag/Nb fusion can mediate specific binding between *E. coli* and effectively promote biofilm formation.
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| Figure 4. Biofilm Growth at 0, 2, and 5.5 Hours.
Images were captured under a 150x objective lens in brightfield and fluorescence. |
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| Figure 5. Visualization of Biofilm Formation through Microscopy Imaging.
Magnification: 150x Video Duration: Captured at 5 min intervals |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1305
Illegal BsaI.rc site found at 2201
- ↑ Piñero-Lambea, C., Bodelón, G., Fernández-Periáñez, R., Cuesta, A. M., Álvarez-Vallina, L., & Fernández, L. Á. (2015). Programming controlled adhesion of E. coli to target surfaces, cells, and tumors with synthetic adhesins. ACS Synthetic Biology, 4(4), 463–473. https://doi.org/10.1021/sb500252a
- ↑ 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
- ↑ Kim, H., Skinner, D. J., Glass, D. S., Hamby, A. E., Stuart, B. A. R., Dunkel, J., & Riedel-Kruse, I. H. (2022). 4-bit adhesion logic enables universal multicellular interface patterning. Nature, 608(7922), 324–329. https://doi.org/10.1038/s41586-022-04944-2
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