Designed by: Shinjini Saha Group: iGEM14_MIT (2014-10-17)
pENTR_BACE1
Protein found in human brain. Shown to cause mis-cleave amyloid-precursor-protein, increasing beta-amyloid formation.
Sequence and Features
Assembly Compatibility:
10
INCOMPATIBLE WITH RFC[10]
Illegal PstI site found at 637 Illegal PstI site found at 911
12
INCOMPATIBLE WITH RFC[12]
Illegal PstI site found at 637 Illegal PstI site found at 911
21
COMPATIBLE WITH RFC[21]
23
INCOMPATIBLE WITH RFC[23]
Illegal PstI site found at 637 Illegal PstI site found at 911
25
INCOMPATIBLE WITH RFC[25]
Illegal PstI site found at 637 Illegal PstI site found at 911
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 136 Illegal SapI.rc site found at 145
The first experiment was based on Anaspec’s SensoLyte 520 β-Secretase Assay Kit. This commercial kit uses a FRET-based technique wherein biological samples are mixed with a proprietary solution containing a BACE1/2 mimic substrate. One terminal of this mimic substrate is conjugated to a fluorophore and the other terminal is conjugated to a fluorescence quencher. So proteolytic Bace1/2 activity causes cleavage of the proprietary substrate, which increases fluorescence due to separation of the fluorophore from the quencher. Relative Bace1 activity in biological samples can be assayed by measuring the intensity of the fluorophore emission and comparing to a standard curve.
miRNA Generator 1
miRNA Generator 2/b>
miRNA Generator 3
miRNA Generator 4
Determining miRNA efficacy in down-regulating BACE1 expression. 30,000,000 cells from each sample were analyzed. RFU represents "relative fluorescence units", a proxy for determining Bace1 protease activity. The shown fluorescence values represent RFU after background fluorescence (as determinined by substrate-only control samples) was subtracted from raw output.
