Part:BBa_K4907136

I0500-B0034-mv140-linker-his tag-B0015

Biology

MV¹⁴⁰

MipA, a surface display protein that can anchor to the membrane surface of E. coli, belongs to the MipA/OmpV family. The current study shows that MipA is expressed and functions in strains of both E. coli K12 and B strains. (1) Through the structural analysis, the MipA protein contains five extracellular loops that form a β-sheet protruding from the cell surface. Among these loops, the third, fourth and fifth loops are primarily considered, since they likely have stronger and more stable anchoring ability in the β-barrel structure of E. coli. Therefore, to better exert MipA activity, we chose MV¹⁴⁰, a derivative of MipA as the surface display protein. MV¹⁴⁰truncated the nucleotide at position 140 at the C-terminus of MipA, which showed higher surface display efficiency compared with MipA.

Usage and design

Anchoring of cellulose-binding protein to the bacterial surface is an important part of the NAIADS project. Based on 2021 (SALVAGE) and 2022 (OMEGA) project of XMU-China, we further refined the bacterial surface display system. We chose MV¹⁴⁰ as the surface display protein which will connect the cellulose binding protein (BBa_K4907027) on the bacterial surface. Cellulose-binding protein will bind to the cellulose of plant roots, thus allowing the engineered bacteria to stick to and perform functions around the roots.

To verify the surface display ability of MV¹⁴⁰, we added the his tag (6×his) after it and tested the fluorescence intensity by immunofluorescence, and analyzed whether MV¹⁴⁰ could be successfully expressed and displayed on the bacterial surface.

Fig. 1 Graphic description of the MV¹⁴⁰ surface display system in NAIADS project.

Characterization

Agarose gel electrophoresis (AGE)

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (2188 bp) can be observed at the position between 2000 and 3000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907136_pSB1C3.

Ability of MV¹⁴⁰ anchoring on the surface of engineered bacteria

First, we used 2% L-arabinose solution to induce the expression of the surface-display system. Then FITC-labeled anti-His-tag antibody was added to characterize whether the displayed MV¹⁴⁰ itself could bind on the surface of engineered bacteria.

Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria. (p = 0.004608 ).

The ratio of fluorescence intensity (λ_Ex = 492 nm, λ_Em = 518 nm) to OD₆₀₀ of the experimental group (express the surface-display system) is higher than that of negative control (not express the surface-display system) (Fig. 3), which indicates the anchoring ability of MV¹⁴⁰ to the surface of bacteria.

Reference

1. M. J. Han, Novel Bacterial Surface Display System Based on the Escherichia coli Protein MipA. J Microbiol Biotechnol 30, 1097-1103 (2020). Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961