Difference between revisions of "Part:BBa M50101:Experience"
| Line 5: | Line 5: | ||
===Applications of BBa_M50101=== | ===Applications of BBa_M50101=== | ||
| − | We used the optogenetic transcription factor EL-222 to drive transcription of | + | We used the optogenetic transcription factor EL-222 to drive transcription of another construct. We attached a p2A DasherGFP at the end of the other construct to infer transcription/expression activity. |
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. | ||
| Line 29: | Line 29: | ||
Grey - Mock, Red - No Light, Purple - 24 hr light (20s on, 60s off) | Grey - Mock, Red - No Light, Purple - 24 hr light (20s on, 60s off) | ||
| + | |||
| + | After 24 hrs of lighting, we were able to observe low efficiency GFP expression in dual transfected groups, but no in any of the controls. | ||
| + | |||
| + | |||
| + | |||
===User Reviews=== | ===User Reviews=== | ||
<!-- DON'T DELETE --><partinfo>BBa_M50101 StartReviews</partinfo> | <!-- DON'T DELETE --><partinfo>BBa_M50101 StartReviews</partinfo> | ||
Revision as of 23:35, 13 December 2017
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_M50101
We used the optogenetic transcription factor EL-222 to drive transcription of another construct. We attached a p2A DasherGFP at the end of the other construct to infer transcription/expression activity.
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.
From top to bottom: dual transfection (top), c120 only transfection (middle), el222 only (bottom)
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.
Grey - Mock, Red - No Light, Purple - 24 hr light (20s on, 60s off)
After 24 hrs of lighting, we were able to observe low efficiency GFP expression in dual transfected groups, but no in any of the controls.
User Reviews
UNIQ57ca270db600aa6d-partinfo-00000000-QINU UNIQ57ca270db600aa6d-partinfo-00000001-QINU