Difference between revisions of "Part:BBa M50101:Experience"
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Cells were transfected with these DNA constructs using Lipofectamine 3000 Reagent, Opti-MEM media, and P3000 (Thermo Fisher). Dual transfections with a maximum of 2μg of total DNA were added to each well at varying ratios of EL222 to C120-Insulin. Controls included single single transfections of EL222, C120-Insulin, GFP positive control (pHela), and a mock transfection. 24 hours post-transfection, fresh DMEM media was added. Effective transfection of pVP-EL222 was confirmed via microscopy. Light stimulation was then initiated for 24 hrs (465 nm, 8W/m^2,20s on-60s off continuous loop). Cells were kept at 37℃ and 5% CO2 during illumination. | Cells were transfected with these DNA constructs using Lipofectamine 3000 Reagent, Opti-MEM media, and P3000 (Thermo Fisher). Dual transfections with a maximum of 2μg of total DNA were added to each well at varying ratios of EL222 to C120-Insulin. Controls included single single transfections of EL222, C120-Insulin, GFP positive control (pHela), and a mock transfection. 24 hours post-transfection, fresh DMEM media was added. Effective transfection of pVP-EL222 was confirmed via microscopy. Light stimulation was then initiated for 24 hrs (465 nm, 8W/m^2,20s on-60s off continuous loop). Cells were kept at 37℃ and 5% CO2 during illumination. | ||
| − | We observed strong RFP expression after only one day post-transfection using 1.5 ug of EL222 in our transfection. RFP was observed in dual transfection (blue and purple) and EL-222 only (red) cells but not in c120 only (green) or mock (grey) transfection groups. | + | We observed strong RFP expression in any cells with EL222 plasmid after only one day post-transfection using 1.5 ug of EL222 in our transfection. |
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| + | [[File:RFPmic1.png]] | ||
| + | [[File:RFPmic2.png]] | ||
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| + | RFP expression was retained two days post-transfection, as shown by flow-cytometry data. RFP was observed in dual transfection (blue and purple) and EL-222 only (red) cells but not in c120 only (green) or mock (grey) transfection groups. | ||
[[File:RFPdual.png]] | [[File:RFPdual.png]] | ||
[[File:RFPexp.png]] | [[File:RFPexp.png]] | ||
Y-axis: Normalized Count X-axis: RFP fluorescence | Y-axis: Normalized Count X-axis: RFP fluorescence | ||
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Using lower amounts (> 1 ug) of EL222 in transfection may lead to very low transfection efficiency, although more rigorous testing is required to prove this claim. | Using lower amounts (> 1 ug) of EL222 in transfection may lead to very low transfection efficiency, although more rigorous testing is required to prove this claim. | ||
Revision as of 23:29, 13 December 2017
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Applications of BBa_M50101
We used the optogenetic transcription factor EL-222 to drive transcription of this construct. We also attached a p2A DasherGFP at the end of this construct to infer transcription/expression activity. Note that the preproinsulin was modified to include a 6xHis Tag.
Cells were transfected with these DNA constructs using Lipofectamine 3000 Reagent, Opti-MEM media, and P3000 (Thermo Fisher). Dual transfections with a maximum of 2μg of total DNA were added to each well at varying ratios of EL222 to C120-Insulin. Controls included single single transfections of EL222, C120-Insulin, GFP positive control (pHela), and a mock transfection. 24 hours post-transfection, fresh DMEM media was added. Effective transfection of pVP-EL222 was confirmed via microscopy. Light stimulation was then initiated for 24 hrs (465 nm, 8W/m^2,20s on-60s off continuous loop). Cells were kept at 37℃ and 5% CO2 during illumination.
We observed strong RFP expression in any cells with EL222 plasmid after only one day post-transfection using 1.5 ug of EL222 in our transfection.
RFP expression was retained two days post-transfection, as shown by flow-cytometry data. RFP was observed in dual transfection (blue and purple) and EL-222 only (red) cells but not in c120 only (green) or mock (grey) transfection groups.
Y-axis: Normalized Count X-axis: RFP fluorescence
Using lower amounts (> 1 ug) of EL222 in transfection may lead to very low transfection efficiency, although more rigorous testing is required to prove this claim.
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