Part:BBa_K4765121
ribozyme connected: galU + pgmA + mScarlet
Contents
Introduction
This composite part is designed for the production of exopolysaccharide (EPS), and 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 EPS biosynthesis pathway [1]. 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 FormingWe 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.
Adhesion AssayTo 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.
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.
Sequence and Features
Assembly Compatibility:
References
|
| proteins |