Difference between revisions of "Part:BBa K4907028"

Latest revision as of 09:28, 11 October 2023

mv140-linker-his tag

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) By structural analysis of it, 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.

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 stick to and perform functions around the roots. This part is meant to express MV¹⁴⁰ with his-tag(6×his) under control of L-arabinose-inducible promoter, thus we can verify the surface display ability of MV¹⁴⁰by immunofluorescence. We added L-arabinose-induced promoter I0500, ribosome binding site and terminator B0015 to basic part to create a composite part (BBa_K4907136).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 transformants were correct. Target bands (2188 bp) can be observed at the position between 2000 and 3000 bp (Fig. 1).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4907136_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).

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
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4907028 short</partinfo>
 ===Biology===
-MV<sup>140</sup>
+====MV<sup>140</sup>====
 MipA, a surface display protein that can anchor to the membrane surface of <i>E. coli</i>, belongs to the MipA/OmpV family. The current study shows that MipA is expressed and functions in strains of both <i>E. coli</i> K12 and B strains. (1) By structural analysis of it, 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 <i>E. coli</i>. Therefore, to better exert MipA activity, we chose MV<sup>140</sup>, a derivative of MipA as the surface display protein. MV<sup>140</sup> truncated 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 ([https://2021.igem.org/Team:XMU-China SALAGE]) and 2022 ([https://2022.igem.wiki/xmu-china/index.html OMEGA]) project of XMU-China, we further refined the bacterial surface display system. We chose MV<sup>140</sup> as the surface display protein which will connect the cellulose binding protein (<partinfo>BBa_K4907027</partinfo>) 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.
 This part is meant to express MV<sup>140</sup> with his-tag(6×his) under control of <i>L</i>-arabinose-inducible promoter, thus we can verify the surface display ability of MV<sup>140</sup>by immunofluorescence.
-We added <i>L</i>-arabinose-induced promoter I0500, ribosome binding site and terminator B0015 to basic part to create a composite part (BBa _ K4907136).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.
+We added <i>L</i>-arabinose-induced promoter I0500, ribosome binding site and terminator B0015 to basic part to create a composite part (<partinfo>BBa_K4907136</partinfo>).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===
@@ Line 15: / Line 16: @@
 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. 1).
-<b>Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4907136_pSB1C3.</b>
+<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/wei-hu/bba-k49071361.png" width="400px"></html></center>
+<center><b>Fig. 1 DNA gel electrophoresis of the colony PCR products of <partinfo>BBa_K4907136</partinfo>_pSB1C3.</b></center>
 ===Reference===
-.	M. J. Han, Novel Bacterial Surface Display System Based on the Escherichia coli Protein MipA. J Microbiol Biotechnol <b>30</b>, 1097-1103 (2020).
+.	M. J. Han, Novel Bacterial Surface Display System Based on the <i>Escherichia coli</i> Protein MipA. <i>J Microbiol Biotechnol</i> <b>30</b>, 1097-1103 (2020).