Difference between revisions of "Part:BBa K4907029"

Revision as of 06:29, 5 October 2023

mv140-linker-cbm-his tag

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

Anchoring of cellulose-binding protein to the bacterial surface is an important part of the NAIADS project. Based on 2021 (SALAGE) 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 adsorb and perform functions around the roots.

This part is meant to express a fusion protein of MV¹⁴⁰ and CBM with his-tag(6×his) under control of L-arabinose-inducible promoter, thus we can verify that CBM could be successfully expressed and displayed on the bacterial surface by immunofluorescence.

We added L-arabinose-induced promoter I0500, ribosome binding site and terminator B0015 to basic part to create a composite part (BBa _ K4907137).Then,the composite part is assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmid was transformed into DH10β, then the positive transformants were selected by chloromycetin and confirmed by colony PCR and sequencing.

Characterization

Agarose gel electrophoresis (AGE)

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformatants 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_K490713_pSB1C3.

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