Part:BBa_K2751012
ALB signal peptide fused mCherry
Usage and Biology
This is a part improved from part: BBa_J06504, which is monomeric RFP optimized for bacteria, aka mCherry.
Albumin (ALB) signal peptide can be tagged to protein of interest and allow the protein of interest to be secreted extracellularly. This method has been proved by C. Attallah et al[1] . We improved the part BBa_J06504 by adding to the 5' end of it a secreting signal peptide (ALB). This design enables us to detect mCherry protein extracellularly. mCherry is a red fluorescent protein that matures rapidly and is widely used as a reporter. We hope that we would be able to detect the extracellular mCherry reporter via a plate/fluorescent reader.
Characterization
Length confirmation: The ALB-mCherry insert is 793bp, and the estimated length including a biobrick prefix is 825bp.
Functionality Test
We transfected HEK293T cells via the GenJet Plus DNA transfection reagent and then collected cell supernatant for fluorescence measurement after 24 and 48 hours. Transparent alpha-MEM medium was used to minimize background noise. We used the Varioskan LUX spectral scanning multimode reader to measure fluorescence levels. Excitation wavelength was 587nm, emission wavelength was 610nm, and bandwidth was 5nm. Result is shown in the figure below. Cultured cell control is taken from the supernatant of cells that have not been transfected with the internal control plasmid.
Reference: 1.A highly efficient modified human serum albumin signal peptide to secrete proteins in cells derived from different mammalian species. (2017, January 10). Retrieved from https://www.sciencedirect.com/science/article/pii/S1046592816305034
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 771
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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