Part:BBa_K5048047

Neae-FLAG

Codes for the surface presentation system containing an export tag, a LysM sequence, a β-barrel, and a Spacer sequence. Codon optimized for expression in E.coli(Fig 1), and a FLAG tag was added for convenient detection.

Fig 1 The overview for Neae

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1244
  

Design

This is a surface display system that we used to present our adhesins LAP and HSP60 on the outer membrane of E. coli. Condon optimized for better expression in our chassis E.coli. Cooperated with HSP60 and LAP, we expected to achieve cell-cell adhesion and cell-intestine adhesion in the small intestine(Fig 2).

Fig 2 The expected effect for the Adhesion module, which adheres to the engineering bacteria and intestine

Source

Original sequence found in the paper:Glass DS, Riedel-Kruse IH. A Synthetic Bacterial Cell-Cell Adhesion Toolbox for Programming Multicellular Morphologies and Patterns. Cell. 2018 Jul 26;174(3):649-658.e16. doi: 10.1016/j.cell.2018.06.041. Epub 2018 Jul 19. PMID: 30033369. UniProt：H3JUY6. We used the N-terminal domain of the protein.

Experiments

This part is expressed on the outer membrane of E. coli BL21(DE3), and we conducted a range of experiments to confirm the presentation of DNA and protein. We also cloned it onto the Neae-HSP60 plasmid(BBa_K5048069) and Neae-LAP plasmid(BBa_K5048068).

The overall experiments we conducted on BBa_K5048047 include:

1. Verification of transformation of plasmid Neae-FLAG
2. Verification of protein Neae
3. Verification of protein Neae on the outer membrane
4. Clone Neae onto improvement parts

Result

Verification of transformation

To evaluate the functions of the target proteins, we transform the pET-28a-[Neae-Flag] into strain E. coli BL21(DE3). We conducted colony PCR to verify the transformation(Fig 3). Moreover, The Sanger sequencing conducted by the cooperated company ensured the specific sequence (Fig 4).

Fig 3 The confirmation of transformation.
Neae 3 is a successful transcription sample

Fig 4 The sequencing result for transformation in ''E. coli'' BL21(DE3).
C: The sequencing result for pET-28a-[Neae-Flag]. The base deletion at the end is due to the limitations of the Sanger method, rather than a base mutation.

Verification of protein Neae

We utilize Western Blot (WB)(Fig 5) to detect the Neae protein.

Fig 5 Western blot analysis for Neae protein.
C: WB analysis of Nese protein in E. coli BL21(DE3) under induction of 0.5mM IPTG and 200rpm for 6.5 hours. The differences in induction temperatures are marked on the image. The exposure time is 20 seconds.

Verification of protein Neae on the outer membrane

To determine whether the display protein, Neae, anchors on the outer membrane or not, we conducted two experiments.

Firstly, we employ indirect immunofluorescence(IF) to confirm the appearance of Neae. The result demonstrates that Neae perform better at 30℃ with a stronger fluorescence(Fig 6).

Fig 6 indirect immunofluorescence for Neae on the outer membrane in E. coli BL21(DE3). Primary antibody: Mouse anti DDDDK-Tag (mAb); secondary antibody: 594-conjugated Goat anti-Rabbit lgG(H+L). The Neae can be visualized in red at an excitation wavelength of 585nm, and the DNA in blue (DAPI) at an excitation wavelength of 358nm. The cells were under an induction of 0.5mM IPTG for 2 hours.

Furthermore, we also conducted WB to ensure the expression level of Neae on the outer membrane(Fig 6). The result indicates that Neae is successfully present on the outer membrane as well and yields more at a higher temperature, which corresponds to the IF result(Fig 7).

Fig 7 WB analysis of Neae protein in ''E. coli.'' BL21(DE3) under the induction of 0.5mM IPTG and 250rpm for 3 hours. The differences in induction temperatures are marked on the image. The exposure time is 10 seconds..

Clone Neae onto improved parts

See more in Neae-HSP60 plasmid(BBa_K5048069) and Neae-LAP plasmid(BBa_K5048068).

Applications of BBa_K5048047

Cooperating with protein LAP and HSP60, we make two kinds of engineered bacteria adhere to each other. (Fig 8)

Fig 8 The expected effect for Adhesion construct