Part:BBa_K4765121

ribozyme connected: galU + pgmA + mScarlet

Introduction

This composite part is designed for the production of exopolysaccharide (EPS), and the detection of EPS’s adhesion ability. It includes E. coli galU on the upstream site and E. coli pgmA on the downstream site, two essential enzymes in the EPS biosynthesis pathway . These two enzymes were tested by iGEM20_XJTU-China in K3331001(galU)and K3331002(pgmA). The following mScarlet is utilized to detect E. coli 's adhesion ability. Additionally, during the validation of EPS, we observed that EPS-expressing bacteria became "heavier" and "stickier", thereby promoting cluster formation.

Usage and Biology

We use this part to improve EPS production of E. coli and assessment of EPS’s adhesion ability.

Characterization

Agarose gel electrophoresis

Figure 1. Agarose gel electrophoresis of PCR products, amplified from bacterial colonies/cultures. From left lane(1) to right lane(3) indicate the successful construction of galU, galU + pgmA, and galU + pgmA + mScarlet.

Successful Protein Expression

Figure 2. SDS-PAGE electrophoresis of BBa_K4765121 and BBa_K4765122 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 are "heavier" and precipitate faster, likely due to being 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 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 Results The 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 being loaded 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 effectively 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 fluorescence 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 Assembly Compatibility: 10 COMPATIBLE WITH RFC[10] 12 INCOMPATIBLE WITH RFC[12] Illegal NotI site found at 3285 21 COMPATIBLE WITH RFC[21] 23 COMPATIBLE WITH RFC[23] 25 COMPATIBLE WITH RFC[25] 1000 COMPATIBLE WITH RFC[1000] References