Difference between revisions of "Part:BBa K3598012"
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The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br. | The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
− | + | [[File:T--BEIJING 4ELEVEN--Acf151 1.png|600px|thumb|center|Figure 1. Part demonstration]] | |
+ | Our AOX1-csgA-mfp5-mfp5 is a composite part consisting of an AOX1 Promoter, an csgA-mfp5-mfp5 recombinant protein sequence, and an AOX1 Terminator. It regulates the expression of our recombinant cohesive protein. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression. Then we verified the production of adhesive proteins by SDS-PAGE electrophoresis. However, the bands in the results are larger than they should be. This might be because that plasmids are integrated into the yeast genome with multiple copies, producing proteins that fold together. | ||
+ | [[File:T--BEIJING 4ELEVEN--Acf 2.png|600px|thumb|center|Figure 2. SDS-PAGE gel analysis of supernatant samples during csgA-mfp5-mfp5 fermentation]] | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> |
Revision as of 08:07, 25 October 2020
AOX1 Promoter_CsgA_mfp5_mfp5_AOX1 Terminator
The circuit we transformed into Pichia Pastoris to produce AMP CEN1HC-Br.
Usage and Biology
Our AOX1-csgA-mfp5-mfp5 is a composite part consisting of an AOX1 Promoter, an csgA-mfp5-mfp5 recombinant protein sequence, and an AOX1 Terminator. It regulates the expression of our recombinant cohesive protein. We inserted the sequence of the system onto vector pPIC9K and transferred the resulting plasmid into Pichia pastoris inoculated on BMMY culture, then added 5% methanol every day to induce its expression. Then we verified the production of adhesive proteins by SDS-PAGE electrophoresis. However, the bands in the results are larger than they should be. This might be because that plasmids are integrated into the yeast genome with multiple copies, producing proteins that fold together.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 2127
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 937
Illegal XhoI site found at 1191 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2193
- 1000COMPATIBLE WITH RFC[1000]