Difference between revisions of "Part:BBa M50101:Experience"
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===Applications of BBa_M50101===
 
===Applications of BBa_M50101===
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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.  
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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.
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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)
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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.
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[[File:GFP1.png]]
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[[File:GFP2.png]]
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From top to bottom: dual transfection (top), c120 only transfection (middle), el222 only (bottom)
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We conclude that VPEL222 can induce target gene expression, albeit with low efficiency (in this case). Further optimization of plasmid ratios and transfection will be needed to test this conclusion further. A stable cell line would also help tremendously in improving transfection success.
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===Stanford Location===
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Plasmid Name: pVPEL222
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Organism: HEK293T cells
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Type: Sensor
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Barcode #:
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Label: BIOE44 F2017
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===User Reviews===
 
===User Reviews===
 
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<!-- DON'T DELETE --><partinfo>BBa_M50101 StartReviews</partinfo>

Latest revision as of 16:34, 14 December 2017


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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.

RFPmic1.png

RFPmic2.png

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.

RFPdual.png RFPexp.png

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.

RFPlow.png

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.

GFP1.png

GFP2.png

From top to bottom: dual transfection (top), c120 only transfection (middle), el222 only (bottom)

We conclude that VPEL222 can induce target gene expression, albeit with low efficiency (in this case). Further optimization of plasmid ratios and transfection will be needed to test this conclusion further. A stable cell line would also help tremendously in improving transfection success.

Stanford Location

Plasmid Name: pVPEL222

Organism: HEK293T cells

Type: Sensor

Barcode #:

Label: BIOE44 F2017

User Reviews

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