Difference between revisions of "Part:BBa M50102:Experience"

 
 
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===Applications of BBa_M50102===
 
===Applications of BBa_M50102===
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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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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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We observed minimal GFP expression following 6 hrs of blue light exposure according to flow cytometry.
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[[File:GFPnoexp.png]]
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Y-axis: normalized count, X-axis: GFP fluorescence
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 +
dual transfection (blue and purple), mock (grey)
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 +
[[File:GFPnoexp1.png]]
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 +
Y-axis: normalized count, X-axis: GFP fluorescence
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 +
EL-222 only (red), c120 only (green), mock (grey)
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 +
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We were able to observe GFP from this construct after 24 hrs of blue light stimulation, although expression was relatively inefficient.
 +
 +
[[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 think that the limited GFP expression efficiency may have resulted from only using 3 c120 repeats instead of 5. This would have lowered the binding affinity of the EL222 protein to the c120 repeat site. It could also be likely that we weren't using enough c120 plasmid in our transfection - we tested varying amounts of c120 plasmid.
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 +
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GFP is leaky at relatively high amounts of this construct (> 1ug DNA) following 24 hrs of light.
 +
 +
[[File:GFPleak1.png]]
 +
 +
Y-axis: normalized count, X-axis: GFP fluorescence
 +
 +
24hr light - 1 ug c120 (purple), no light - 1 ug c120 (red), mock(grey)
 +
 +
[[File:GFPleak2.png]]
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 +
Y-axis: normalized count, X-axis: GFP fluorescence
 +
 +
24hr light - 1 ug c120 (orange), no light - 1 ug c120 (orange), mock(red)
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The efficiency of this system still needs optimization, but preliminary results show some level of ability to drive gene expression using light.
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===Stanford Location===
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Plasmid Name: pc120-Insulin
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Organism: HEK293T cells
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Type: Actuator
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Barcode #: 133011794
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Label: BIOE44 F2017
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 16:40, 14 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_M50102

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 minimal GFP expression following 6 hrs of blue light exposure according to flow cytometry.

GFPnoexp.png

Y-axis: normalized count, X-axis: GFP fluorescence

dual transfection (blue and purple), mock (grey)

GFPnoexp1.png

Y-axis: normalized count, X-axis: GFP fluorescence

EL-222 only (red), c120 only (green), mock (grey)


We were able to observe GFP from this construct after 24 hrs of blue light stimulation, although expression was relatively inefficient.

GFP1.png

GFP2.png

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

We think that the limited GFP expression efficiency may have resulted from only using 3 c120 repeats instead of 5. This would have lowered the binding affinity of the EL222 protein to the c120 repeat site. It could also be likely that we weren't using enough c120 plasmid in our transfection - we tested varying amounts of c120 plasmid.


GFP is leaky at relatively high amounts of this construct (> 1ug DNA) following 24 hrs of light.

GFPleak1.png

Y-axis: normalized count, X-axis: GFP fluorescence

24hr light - 1 ug c120 (purple), no light - 1 ug c120 (red), mock(grey)

GFPleak2.png

Y-axis: normalized count, X-axis: GFP fluorescence

24hr light - 1 ug c120 (orange), no light - 1 ug c120 (orange), mock(red)

The efficiency of this system still needs optimization, but preliminary results show some level of ability to drive gene expression using light.

Stanford Location

Plasmid Name: pc120-Insulin

Organism: HEK293T cells

Type: Actuator

Barcode #: 133011794

Label: BIOE44 F2017

User Reviews

UNIQ7655206ed2e4839c-partinfo-00000000-QINU UNIQ7655206ed2e4839c-partinfo-00000001-QINU