Part:BBa_K4907135

I0500-B0034-cbm-his tag-B0015

Biology

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. MV¹⁴⁰ truncated the nucleotide at position 140 at the C-terminus of MipA, which showed higher surface display efficiency compared with MipA. For cellulose-binding proteins, we chose the cellulose binding domain (CBDcex) of an extracellular glucanase derived from Celluomonas fimi.The literature suggests that CBDcex can be successfully expressed and exert a cellulose-binding function in E. coli JM101.(2)

Usage and design

In order to contrast the function of MV¹⁴⁰ more precisely, this circuit was constructed as a negative control for BBa_K4907137. This circuit does not contain MV¹⁴⁰, so it is impossible to display CBM on the bacterial surface, and the ratio of fluorescence intensity / OD ₆₀₀ will be much lower than that of the experimental group(contain the surface display system).

Fig. 1 Graphic description of the circuit 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 (2235 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_K4907135_pSB1C3.

Ability of binding CBM 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 could bind CBM on the surface of engineered bacteria.

Fig. 3 The results of immunofluorescence to probe the binding event on the surface of engineered bacteria. (p= 0.003578,p-value: no significance (ns), 0.0332 (*), 0.0021 (**), 0.0002 (***), <0.0001 (****). ).

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).

2. E. Ong, N. R. Gilkes, R. C. Miller, Jr., R. A. Warren, D. G. Kilburn, The cellulose-binding domain (CBD(Cex)) of an exoglucanase from Cellulomonas fimi: production in Escherichia coli and characterization of the polypeptide. Biotechnol Bioeng 42, 401-409 (1993).

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
  Illegal AgeI site found at 1504
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961