Difference between revisions of "Part:BBa M36907:Experience"

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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 over time upon exposure to 0-1mT magnetic fields. 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 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.

User Reviews

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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 over time upon exposure to 0-1mT magnetic fields. 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 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.
 ===User Reviews===