Difference between revisions of "Part:BBa K4181014"
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===Usage and Biology=== | ===Usage and Biology=== | ||
<p>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. </p> | <p>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. </p> | ||
| − | [[File:2022-SJTU-Biox-Shanghai-1.png| | + | [[File:2022-SJTU-Biox-Shanghai-1.png|500px]]<br> |
'''Figure 1.''' Electrophoresis results after PCR of cotE and the first PCR electrophoresis results of cotB. The picture indicates a positive result.<br> | '''Figure 1.''' Electrophoresis results after PCR of cotE and the first PCR electrophoresis results of cotB. The picture indicates a positive result.<br> | ||
<p>Next, PCR the cotB gene sequence again, and add 4B4 melanin-binding peptide and the stop codon to the 3' end of this sequence by designing your primers for this PCR.</p> | <p>Next, PCR the cotB gene sequence again, and add 4B4 melanin-binding peptide and the stop codon to the 3' end of this sequence by designing your primers for this PCR.</p> | ||
| − | [[File:2022-SJTU-Biox-Shanghai-2.png| | + | [[File:2022-SJTU-Biox-Shanghai-2.png|500px|]]<br> |
| − | '''Figure | + | '''Figure 2.''' Electrophoresis results of cotB after the second PCR and Dsup-encoded gene after PCR. The picture indicates a positive result.<br> |
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Revision as of 08:53, 12 October 2022
cotE protein DNA sequence
We added a Linker sequence to the end of the cotE protein sequence to facilitate the production of cotE with a further binding peptide to function with the cotB protein.
Usage and Biology
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. Electrophoresis results after PCR of cotE and the first PCR electrophoresis results of cotB. The picture indicates a positive result.
Next, PCR the cotB gene sequence again, and add 4B4 melanin-binding peptide and the stop codon to the 3' end of this sequence by designing your primers for this PCR.
Figure 2. Electrophoresis results of cotB after the second PCR and Dsup-encoded gene after PCR. The picture indicates a positive result.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 493