Difference between revisions of "Part:BBa M36907:Experience"
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| − | Our group set out to re-engineer a non-invasive sensor orthogonal to optogenetics based on magnetic activation (i.e. magnetogenetics). The ''columba livia'' homologues of two iron-sulfur cluster assembly genes (IscA1, IscA2) were codon-optimized (''IDT, Coralville, IA, U.S.A.'') and oligosynthesized into a GFP-fusion construct (''DNA 2.0, Menlo Park, CA, U.S.A.'') for co-transfection into HEK293T cells with a RFP-fusion calcium-indicator plasmid (GCaMP6s, ''Addgene, Douglas Kim Lab''). The translated IscA proteins have been hypothesized to migrate from the nucleus and bind to cellular membranes as magnetoreceptors in eukaryotic organisms (Long et al. 2015). 48 hours after transient co-transfection, we measured fluorescent readout (normalized whole frame and single cell) over time upon exposure to 0-1mT magnetic fields. | + | Our group set out to re-engineer a non-invasive sensor orthogonal to optogenetics based on magnetic activation (i.e. magnetogenetics). The ''columba livia'' homologues of two iron-sulfur cluster assembly genes (IscA1, IscA2) were codon-optimized (''IDT, Coralville, IA, U.S.A.'') and oligosynthesized into a GFP-fusion construct (''DNA 2.0, Menlo Park, CA, U.S.A.'') for co-transfection into HEK293T cells with a RFP-fusion calcium-indicator plasmid (GCaMP6s, ''Addgene, Douglas Kim Lab''). The translated IscA proteins have been hypothesized to migrate from the nucleus and bind to cellular membranes as magnetoreceptors in eukaryotic organisms (Long et al. 2015). 48 hours after transient co-transfection, we measured fluorescent readout (normalized whole frame and single cell) over time upon exposure to 0-1mT magnetic fields. Information about the image analysis techniques can be found by clicking on the left image. |
| − | [[File: | + | [[File:4 MicroscopyImages-updated.png|400px|thumb|left|IscA1 Normalized Whole Frame Fluorescence]] |
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| + | [[File:Isca1normal.png|400px|thumb|right|IscA1 Normalized Whole Frame Fluorescence]] | ||
[[File:IscA1_SingleCell.jpg|400px|thumb|right|IscA1 Single Cell Fluorescence]] | [[File:IscA1_SingleCell.jpg|400px|thumb|right|IscA1 Single Cell Fluorescence]] | ||
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Our pilot study sensor results reveal strong binary control at B-field levels above 0.25mT for the IscA1-based constructs (no significant change in fluorescence was observed for IscA2 or the control upon magnetic exposure), but did not confirm modular control (R=0.87 for fold-change vs. B-field) due to our small sample size in a constrained setting. | Our pilot study sensor results reveal strong binary control at B-field levels above 0.25mT for the IscA1-based constructs (no significant change in fluorescence was observed for IscA2 or the control upon magnetic exposure), but did not confirm modular control (R=0.87 for fold-change vs. B-field) due to our small sample size in a constrained setting. | ||
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Applications of BBa_M36907
Our group set out to re-engineer a non-invasive sensor orthogonal to optogenetics based on magnetic activation (i.e. magnetogenetics). The columba livia homologues of two iron-sulfur cluster assembly genes (IscA1, IscA2) were codon-optimized (IDT, Coralville, IA, U.S.A.) and oligosynthesized into a GFP-fusion construct (DNA 2.0, Menlo Park, CA, U.S.A.) for co-transfection into HEK293T cells with a RFP-fusion calcium-indicator plasmid (GCaMP6s, Addgene, Douglas Kim Lab). The translated IscA proteins have been hypothesized to migrate from the nucleus and bind to cellular membranes as magnetoreceptors in eukaryotic organisms (Long et al. 2015). 48 hours after transient co-transfection, we measured fluorescent readout (normalized whole frame and single cell) over time upon exposure to 0-1mT magnetic fields. Information about the image analysis techniques can be found by clicking on the left image.
Our pilot study sensor results reveal strong binary control at B-field levels above 0.25mT for the IscA1-based constructs (no significant change in fluorescence was observed for IscA2 or the control upon magnetic exposure), but did not confirm modular control (R=0.87 for fold-change vs. B-field) due to our small sample size in a constrained setting.
Nonetheless, this IscA-based magnetogenetic system opens the door for future refinements and applications in noninvasive neuromodulation, CRISPR-Cas9 delivery, and targeted gene therapy.
“Stanford Location” DNA 2.0 Plasmid Name: MTS_Isca1 (Vector: pD669-AR), DNA2.0 Gene #232002, HEK293T mammalian cells, Sensor, Glycerol Stock Barcode #0128151409, Shriram Engineering Center, Box Label: BIOE44 F15 Section 2 --> Original DNA 2.0 strain transferred to Lei Stanley Qi's Lab for further investigation outside classroom setting.
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