Difference between revisions of "Part:BBa K4765106"

Latest revision as of 15:52, 12 October 2023

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

Introduction

We introduced a self-assembly synthetic biofilm formation system by transfecting intimin-Nb3 fusion into E. coli. Intimin-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 OD600 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 OD600 measurement) by the bacterial count at 0 hours.

As is shown in Figure 1 and 2, 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.

As is shown in Figure 3, for aTc-induced intimin-Ag3/Nb3 pairs, bacteria remaining in the supernatant was significantly lower than the uninduced samples at 3 and 6 hours. These results collectively demonstrate that the intimin-Ag/Nb pairs can effectively promote the binding between E. coli.

Figure 1. Bacteria Percentage Remaining in the Supernatant at 3 Hours. The bacterial quantity in the supernatant is quantified by measuring the OD600 (1 OD600 corresponds to 10^8 bacterial particles).

Figure 2. 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.

Figure 3. Bacteria Remaining in the Supernatant at 0,3,6 Hours The bacterial quantity(not-induced/aTc-induced intimin-Ag3/Nb3 bacteria) in the supernatant is quantified by OD600 (1 OD600 corresponds to 10^8 bacterial particles)

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.

Figure 4. Biofilm Growth at 0, 2, and 5.5 Hours. Images were captured under a 150x objective lens in brightfield and fluorescence.

Figure 5. Visualization of Biofilm Formation through Microscopy Imaging. Magnification: 150x Video Duration: Captured at 5 min intervals

Sequence and Features

Reference

1. ↑ 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
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
3. ↑ 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