Difference between revisions of "Part:BBa K4765121"

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To confirm if the ''E. coli'' expressing EPS became more “sticker”, we allowed the bacterial suspension to settle for a period and  
 
To confirm if the ''E. coli'' expressing EPS became more “sticker”, we allowed the bacterial suspension to settle for a period and  
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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.
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observed its aggregation process. As is shown in Figure 3, EPS-expressing ''E. coli'' exhibited significantly faster aggregation compared to the control group, indicating that EPS-expression bacteria is "sticker", leading to bacterial aggregation.
  
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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.
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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 4, 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.
  
 
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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.
 
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.
  
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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.
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As is shown in Figure 5, 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.
  
 
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<span class='h3bb'>Sequence and Features</span>
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===Sequence and Features===
 
<partinfo>BBa_K4765121 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4765121 SequenceAndFeatures</partinfo>
  
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==References==
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===References===
 
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Latest revision as of 15:55, 12 October 2023


ribozyme connected: galU + pgmA + mScarlet contributed by Fudan iGEM 2023


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 [1]. These two enzymes were tested by iGEM20_XJTU-China in BBa_K3331001and BBa_K3331002. 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

contributed by Fudan iGEM 2023
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

contributed by Fudan iGEM 2023
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 3, 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 4, 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.

contributed by Fudan iGEM 2023
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.

contributed by Fudan iGEM 2023
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 5, 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.

contributed by Fudan iGEM 2023
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.

contributed by Fudan iGEM 2023
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

  1. ↑ Levander, F., Svensson, M., & Rådström, P. (2002). Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus. Applied and Environmental Microbiology, 68(2), 784–790. https://doi.org/10.1128/AEM.68.2.784-790.2002