Part:BBa_K245050
GyrB-CutA1
The part is designed to prepare a polypeptide composed of gyrase B fragment on N-terminus and CutA1 on C-terminus. CutA1 is a bacterial protein that binds metal ions and is a trimeric protein. Gyrase B fragment can dimerize in the presence of aminocoumarin antibiotic coumermycin and disassemble by novobiocin. The purpose of this construct is to prepare material that can be assembled or disassembled by the addition of small molecules. In the assembled state the polypeptide can form lattice or aggregates with hexagonal pores.
The part BBa_K245116 codes for the 24 kDa N-terminal GyrB fragment of gyrase subunit B, and the part BBa_K245129 codes for CutA1, a copper binding protein, which are linked by a friendly scar tccggc (SG). Coding sequence for Gyrase subunit B fragment was amplified by PCR from a plasmid. Coding sequence for CutA1 was multiplied by colony PCR from E. coli genomic DNA. Both genes were cloned into the functionalized vector ([1] BBa_K245005) carrying T7 promoter, RBS, ATG at 5' and T7 terminator and STOP codon at 3’ of multiple-cloning site. In this version of vector a sequence coding for His-tag is inserted between ATG and multiple-cloning site, which results in His-tag at the N-terminus of the protein product of the gene. In this way two basic parts were obtained: BBa_K245116 coding for GyrB and BBa_K245129 coding for CutA1.
The basic BioBrick part coding for GyrB (BBa_K245116) was cut with BspEI and PstI restriction enzymes creating back vector and basic part coding for CutA1 (BBa_K245129) was cut with NgoMIV and PstI restriction enzymes creating back insert. Ligation of back vector and back insert resulted in generation of a composite part called GyrB-CutA1 (BBa_K245050, Figure 1), where CutA1 coding sequence is at the 3’ and GyrB coding sequence is at the 5’ of the composite part. The friendly scar between basic parts BBa_K245116 and BBa_K245129 is tccggc (SG). Due to design of our functionalized vector polypeptide product of composite part (BBa_K245050) contains a His-tag on N-terminus.
Figure 1: Scheme of the construct GyrB-CutA1.
E. coli BL21(DE3) pLysS were transformed with BBa_K245050. Protein expression was induced with IPTG. The protein GyrB-CutA1 was isolated from the supernatant of cell lysate (Figure 2). The protein was isolated on a Ni-NTA column. Presence of protein was confirmed by Western blot analysis using anti-His primary antibodies (Figure 3).
Figure 2: Western blot of cell lysate supernatant and pellet.
Figure 3: Western blot of eluted and dialysed protein.
The polypeptide product of this composite part is a combination of a dimerization (GyrB) and a trimerization (CutA1) domain. Assembly of is protein product can be regulated by small chemicals. Addition of aminocoumarin antibiotic coumermycin leads to the dimerization of GyrB domain (Figure 4).
Figure 4: Coumermycin (grey) binds to two gyrase B binding sites (on two gyrase B polypeptide chains coloured green and yellow) so that those two polypeptide chains form a tight dimer.
Because this protein construct is a combination of a dimerization (GyrB) and a trimerization domain (CutA1), assembly of this construct by the addition of coumermycin may result in a hexagonal assembly forming planar lattice or closed assemblies and formation of pores of defined size (Figure 5) This structure can be disassembled by another aminocoumarin antibiotic novobiocin which disrupts coumermycin-induced dimers of GyrB.
Figure 5: Hexagonal packing model of GyrB-CutA1 fusion protein. Left: GyrB dimers are shown in blue, green or yellow color, while the trimerization domain of CutA1 is shown in grey. Right: Hexagonal packing can cover the planar lattice.
Isolated GyrB-CutA1 protein sample cross-linked with coumermycin was examined with transmission electron microscopy (TEM), where we observed formation of a porous material (Figure 6)
Figure 6: Porous structure of the material consisting of GyrB-CutA1 fusion protein after addition of coumermycin seen on TEM. Molecular model of gyrB-CutA1 packing (light blue) is shown in scale for comparison.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 428
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 100
Illegal AgeI site found at 238 - 1000COMPATIBLE WITH RFC[1000]
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