Difference between revisions of "Part:BBa K3610031"
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This part can be used for expressing the plant pattern recognition receptor BRI1-associated receptro kinase (BAK1) from Arabidopsis thaliana in S. cerevisiae. | This part can be used for expressing the plant pattern recognition receptor BRI1-associated receptro kinase (BAK1) from Arabidopsis thaliana in S. cerevisiae. | ||
− | The sequence of the BAK1 protein does not contain the original signal peptide sequence of the receptor. Instead it has been replaced by the secretion signal of the alpha-Factor from S. cerevisiae. | + | The sequence of the BAK1 protein does not contain the original signal peptide sequence of the receptor (BAK-). Instead it has been replaced by the secretion signal of the alpha-Factor from S. cerevisiae. |
To make expression of BAK1 visible and to test observe the localization in the cell, the BAK1 coding region has been fused to a yellow fluorescent protein by a 15 amino-acid long linker. | To make expression of BAK1 visible and to test observe the localization in the cell, the BAK1 coding region has been fused to a yellow fluorescent protein by a 15 amino-acid long linker. | ||
For initiation of the expression the promoter of this part is a truncated version of the ADH1 promoter from S. cerevisiae. For termination this part has terminator sequence of the enolase 2 protein from S. cerevisiae. | For initiation of the expression the promoter of this part is a truncated version of the ADH1 promoter from S. cerevisiae. For termination this part has terminator sequence of the enolase 2 protein from S. cerevisiae. | ||
− | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
+ | |||
+ | ==Characterization== | ||
+ | ===Expression of BAK1-/YFP in S. cerevisiae=== | ||
+ | After successful transformation of yeast cells we checked for expression of the protein under a confocal microscope. Expression of the construct was confirmed. We failed, however, to confirm localization at the cell membrane. | ||
+ | |||
+ | [[File:BAK-.jpg]] | ||
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Revision as of 16:09, 18 October 2020
BAK1 without native signal peptide / YFP
This part can be used for expressing the plant pattern recognition receptor BRI1-associated receptro kinase (BAK1) from Arabidopsis thaliana in S. cerevisiae.
The sequence of the BAK1 protein does not contain the original signal peptide sequence of the receptor (BAK-). Instead it has been replaced by the secretion signal of the alpha-Factor from S. cerevisiae. To make expression of BAK1 visible and to test observe the localization in the cell, the BAK1 coding region has been fused to a yellow fluorescent protein by a 15 amino-acid long linker.
For initiation of the expression the promoter of this part is a truncated version of the ADH1 promoter from S. cerevisiae. For termination this part has terminator sequence of the enolase 2 protein from S. cerevisiae.
Usage and Biology
Characterization
Expression of BAK1-/YFP in S. cerevisiae
After successful transformation of yeast cells we checked for expression of the protein under a confocal microscope. Expression of the construct was confirmed. We failed, however, to confirm localization at the cell membrane.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 1758
Illegal PstI site found at 848
Illegal PstI site found at 893
Illegal PstI site found at 1183 - 1000COMPATIBLE WITH RFC[1000]