Part:BBa_K4181017
A short peptide that binds melanin
4B4 binding peptide can bind melanin, the 4B4 binding peptide produced on the surface of the sporium, we can adsorb more melanin through it, in order to strengthen its resistance to the external harsh environment
Verification of 4B4 Immunofluorescence
cotB and cotE proteins are both innate proteins of B.subtilis which will express when sporulation is induced. They help organize the outer spore surface and therefore can be chosen as the anchoring proteins used in the spore display system, fused with exogeneous proteins to display them on the spore surface. Firstly, since cotB and cotE proteins are bacteriophage surface proteins of Bacillus subtilis and their coding genes are available in their genome, we extracted the genome of Bacillus subtilis strain WB800N, and then used this genome as a template to PCR the gene sequences corresponding to cotE and cotB proteins from the genome. With the design of PCR primers, we added the cotE to the homologous arm of the pHT01 plasmid sequence.A RBS sequence was added to the 5' end of the cotE gene sequence, and the stop codon was removed at its 3' end and the linker sequence was added; the homologous arm of the tyrosinase coding sequence and the RBS sequence were added to the 5' end of the cotB gene sequence, and the stop codon was removed at its 3' end. The stop codon was removed at the 3' end and the FLAG tag was added.
Figure 1. 4B4 detection under immunofluorence microscopy .
From the fig nn, it can be clearly identified the red dots in the middle of the vision in the same place as where the spores were observed by the phase contrast microscopy. Fig nn is the sample with plasmid but no IPTG induced plasmid and figmm is the control group with no plasmid at all. The results revealed that our 4B4 peptide was successfully expressed on the spore surface compared to the nagative controls. In line with the results from tyrosinase, the proportion of spores carrying fluorescence is not so much. The possible reasons and have been illustrated above. Combined with the results we got last time with Alexa Floura 488, it was deduced that the microscopy had obervable attennuation of light in the whole vision, so even if the part around is dark without fluorescence, it could not indicate no fluorenscece existed in those places. This attenuation added difficulties for us to calculate the accurate expression rate so that a flow cytometry assay was conducted later with similar procedures.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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