Difference between revisions of "Part:BBa K4202015"
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We referenced the literature and finally determined the protein expression validation method through extensive experiments. Extracellular secretion and intracellular accumulation of scaffold building blocks by recombinant Bacillus strains was analyzed by preparing four different fractions from cultures for SDS-PAGE analysis. | We referenced the literature and finally determined the protein expression validation method through extensive experiments. Extracellular secretion and intracellular accumulation of scaffold building blocks by recombinant Bacillus strains was analyzed by preparing four different fractions from cultures for SDS-PAGE analysis. | ||
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| − | <div align="center">[[File:YZH-8.png| | + | <div align="center">[[File:YZH-8.png|600px]]</div> |
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| − | <div align="center"><b>Fig 2</b>Experimental workflow used for the analysis of EutM scaffold building block secretion and expression by engineered <i>B. subtilis</i>.</div> | + | <div align="center"><b>Fig 2</b> Experimental workflow used for the analysis of EutM scaffold building block secretion and expression by engineered <i>B. subtilis</i>.</div> |
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| − | <div align="center">[[File:YZH-9.png| | + | <div align="center">[[File:YZH-9.png|600px]]</div> |
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| − | <div align="center"><b>Fig 2</b>SDS-PAGE and Coomassie brilliantblue staining results of EutM-SpyCatcher protein</div> | + | <div align="center"><b>Fig 2</b> SDS-PAGE and Coomassie brilliantblue staining results of EutM-SpyCatcher protein</div> |
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Revision as of 03:48, 1 October 2022
This part is a fusion protein of EutM and SpyCatcher , and it`s used for the bio-scafford
We get the information of the EutM from the BBa_K311004 and other paper. We want to construct a biology scaffold based on this protein and another system SpyCatcher-SpyTag system. Thus, we connect the Spycatcher to the C-terminal of the EutM via a GS linker. In order to enable the protein can be secreted out of the bacterium, we connected the SacB signal sequence of Bacillus subtilis to the N-terminal of EutM. Besides, a His tag is contained between the SacB and EutM for the purification, because the SacB signal sequence will be cleavaged after being secreted. This protein can be utilized with the HagT209C::SpyT588 to form the biological scaffold. This biological scaffold can be used for other area such as purification of sewage, enzyme reaction and so on.
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]
Result:
We obtained the CDS and protein sequence of EutM, engineered SpyCatcher and SacB secretion signal sequence of Bacillus subtilis from NCBI database, and designed the fusion protein SP-EutM-spycatcher. To ensure that the designed fusion protein still has the ability to assemble, I-TASSER was used for homology modeling. The results showed that the recombinant protein could still form a reasonable spatial structure.
We referenced the literature and finally determined the protein expression validation method through extensive experiments. Extracellular secretion and intracellular accumulation of scaffold building blocks by recombinant Bacillus strains was analyzed by preparing four different fractions from cultures for SDS-PAGE analysis.
