Difference between revisions of "Part:BBa K4765121"

From left lane(1) to right lane(3) indicate the successful construction of galU, galU + pgmA, and galU + pgmA + mScarlet.

We constructed BBa_K4765121 and BBa_K4765122 into the pET28a plasmid and transformed it into E. coli BL21 DE3. Lane 1 to 2 represent the protein expression of BBa_K4765121, Lane 3 to 4 represent the protein expression of BBa_K4765122. Each part has one leaky expression version and one induced version. As indicated by the red arrow, we successfully expressed proteins in BBa_K4765121 and BBa_K4765122.

Aggregation Assay and Cluster Forming

We found EPS expressing bacteria is "heavier", precipitate faster, likely due to more "sticky".

To confirm if the *E. coli* expressing EPS became more “sticker”, we allowed the bacterial suspension to settle for a period and observed its aggregation process. As is shown in Figure 2, EPS-expressing E. coli exhibited significantly faster aggregation compared to the control group, indicating that EPS expression bacteria is "sticker", leading to bacterial aggregation.

Additionally，We mixed the *E. coli* expressing EPS with bacteria only expressing StayGold before loading into the flow chamber, and conducted fluorescence microscopy imaging subsequently. As is shown in Figure 3, Bacteria expressing EPS, indicated by red fluorescence, tend to cluster together, while the control group bacteria with green fluorescence are sparsely distributed. This suggests that EPS-expressing bacteria are "stickier" and have the capability to form clusters.

Figure 3. Aggregation Experiment ResultsThe four on the left are EPS-expressing *E. coli*, while the yellow one on the right represents the control group with *E. coli* only expressing stayGold.

Figure 4. Fluorescence Microscopy Imaging (150x) of Cluster Forming Red fluorescence： EPS-expressing E. coli, Green fluorescence：E. coli only expresses StayGold.

Adhesion Assay

To validate the adhesion effects of EPS, we performed microscopy imaging using a chamber-based approach. After mixing the *E.coli* expressing EPS with an appropriate concentration of PDL (Poly-D-Lysine), it was forcefully pipetted ten times before loading into the flow chamber. Subsequently, we conducted fluorescence microscopy imaging. We applied two different force intensities to wash the adherent bacteria and observed whether the EPS-expressing *E.coli* could remain adhered to the glass slide without being dislodged.

As is shown in Figure 4, the two left images under lower-speed washing, EPS-expressing *E. coli* cells (red fluorescence indicated by white arrows) maintained adhesion to the glass surface for an extended period without being dislodged, while the control group (green fluorescence) was washed away. In the two right images, subjecting adherent *E. coli* to more intense washing removed non-adherent cells completely. However, EPS-expressing *E. coli* cells (white arrows) could still adhere to the glass surface for a considerable time. This indicates that EPS effectly promotes *E. coli* adhesion.

Figure 5. Fluorescence Microscopy Imaging (150x) of E.coli Adhesion All four images were captured from the same field of view, showing *E.coli* adhesion under different washing conditions. The two images on the left depict bacterial adhesion over an extended period under mild washing, while the two images on the right show bacterial adhesion still remains under stronger washing.

In the following video, we further demonstrated the adhesion process of EPS-expressing E. coli cells (red fulorescence indicated by white arrows) during the aforementioned washing procedures.

Figure 6. Visualization of *Escherichia coli* Adhesion through Fluorescence Microscopy Magnification: 150x Video Duration: Captured at 200ms intervals

Sequence and Features

References

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