Difference between revisions of "Part:BBa K1391016"
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| + | <h3> hEF1a in the context of MIT iGEM 2014 </h3> | ||
To test our miRNA’s effectiveness at down-regulating BACE1 expression we transfected separate HEK293 cultures with either (1) BACE1 under constitutive expression by the Hef1a promoter or (2) with BACE1 under constitutive expression by the Hef1a promoter and with a miRNA-generating vector inducibly regulated by the TRE promoter. We performed these transfections in duplicate—once with native BACE1, and then once with eYFP-tagged BACE1. There were two variants of this eYFP-tagged BACE1 construct—one with eYFP linked to the N-terminus of Bace1 and another with eYFP on the C-terminus. We created these two variants after considering the possibility that linking eYFP to either terminus of Bace1 might interfere with the peptide’s native structure or function. <br /> <br /> | To test our miRNA’s effectiveness at down-regulating BACE1 expression we transfected separate HEK293 cultures with either (1) BACE1 under constitutive expression by the Hef1a promoter or (2) with BACE1 under constitutive expression by the Hef1a promoter and with a miRNA-generating vector inducibly regulated by the TRE promoter. We performed these transfections in duplicate—once with native BACE1, and then once with eYFP-tagged BACE1. There were two variants of this eYFP-tagged BACE1 construct—one with eYFP linked to the N-terminus of Bace1 and another with eYFP on the C-terminus. We created these two variants after considering the possibility that linking eYFP to either terminus of Bace1 might interfere with the peptide’s native structure or function. <br /> <br /> | ||
Revision as of 21:51, 1 November 2014
pENTR_hEF1a
Strong constitutive promoter for mammalian gene transcription. This part is a repeat of BBa_K779200 .
Sequence and Features
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 7
Illegal EcoRI site found at 1211
Illegal PstI site found at 338
Illegal PstI site found at 843 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 7
Illegal EcoRI site found at 1211
Illegal PstI site found at 338
Illegal PstI site found at 843 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 7
Illegal EcoRI site found at 1211
Illegal BglII site found at 592
Illegal BamHI site found at 1198
Illegal XhoI site found at 991 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 7
Illegal EcoRI site found at 1211
Illegal PstI site found at 338
Illegal PstI site found at 843 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 7
Illegal EcoRI site found at 1211
Illegal PstI site found at 338
Illegal PstI site found at 843
Illegal NgoMIV site found at 726
Illegal AgeI site found at 104 - 1000COMPATIBLE WITH RFC[1000]
hEF1a in the context of MIT iGEM 2014To test our miRNA’s effectiveness at down-regulating BACE1 expression we transfected separate HEK293 cultures with either (1) BACE1 under constitutive expression by the Hef1a promoter or (2) with BACE1 under constitutive expression by the Hef1a promoter and with a miRNA-generating vector inducibly regulated by the TRE promoter. We performed these transfections in duplicate—once with native BACE1, and then once with eYFP-tagged BACE1. There were two variants of this eYFP-tagged BACE1 construct—one with eYFP linked to the N-terminus of Bace1 and another with eYFP on the C-terminus. We created these two variants after considering the possibility that linking eYFP to either terminus of Bace1 might interfere with the peptide’s native structure or function.After transfection and Doxycycline-induction of our miRNA-generating vectors, we used flow cytometry to verify that the miRNA-generating vectors were being expressed. Proper splicing of the mature miRNA out of the expression vector leaves an intact mKate coding sequence in the vector, so emission of red fluorescence from our transfected HEK293 indicates proper miRNA processing.
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