Part:BBa_K2833017

J23104+B0032+intimin+mEos+Ter

Intimin is a virulence factor (adhesin) of EPEC and EHEC E. coli strains, which is expressed on the bacterial cell surface where it can bind to its receptor Tir (Translocated intimin receptor). Intimin are integrated into the bacterial outer membrane with the amino-terminal region, while the carboxy-terminal region of the polypeptide is surface exposed^[1]. So, protein fused to the C-terminal of intimin can be display on the OM of the bacteria.

We linked a fluorescent protein mEos(BBa_K2833003)to the C-terminal of autotransporter to visualize the surface display.

Cell lysis result

We conducted a cell lysis experiment to observe the distribution of fluorescent protein(figure 1). From this result we can see that after cell lysis followed by centrifuge, both the precipitation and supernatant shows relative strong fluorescence signal in intracellular expressing mEos cells(figure 1B, middle), while the signal was stronger in supernatant than in precipitation in mEos surface displayed cells(figure 1B, right). To further confirm this result, we removed the supernatant and observed that the signal in mEos surface displayed cells fragments was stronger than the mEos intracellular expressed ones(figure 1C).This suggests that the surface displayed mEos were anchored to the outer membrane and being precipitated with the cell fragment, while the intracellular expressed mEos were solved in the supernatant.

Fig.1 the fluorescence of intracellular expression of mEos and surface display of mEos via intimin before and after cell lysis. A: before cell lysis; B: after cell lysis

Microscopy results

We observed the fluorescence under the super-resolution microscope. As figure 2 shows, The intracellular expression of mEos displayed the rode-shape of E.coli, while the signal of surface displayed mEos showed the dotted pattern around the cells, which suggested that the mEos were gathered and distributed at the surface of E.coli..

Fig.2 super-resolution microscopy of intimin-mEos Left: intracellular expression of mEos; Right: surface display of mEos via intimin

Reference

[1] Thorsten M. Adams, et al. Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation. OURNAL OF BACTERIOLOGY(2005) p. 522–533

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 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 1306