Part:BBa_K2751013

pCMV-ALB-mEGFP internal control

Usage and Biology

This part contains the pCMV-ALB-mEGFP sequence, and is used as our internal control plasmid. The CMV promoter allows mEGFP to be expressed constantly when transfected into cells. Moreover, the signal peptide (ALB) allows mEGFP to be secreted extracellularly, so that signal detection can become much easier.

Characterization

We ran electrophoresis to check if the length of the part is correct. The length of CMV promoter, ALB sequence, and mEGFP sequence are 290bp, 56bp, and 720bp respectively. Therefore, we expect a product of 1066bp (1099bp along with a biobrick prefix). Gel photo is shown in the figure below.

Functionality Test

We transfected HEK293T cells via the GenJet Plus DNA transfection reagent and then collected cell supernatant for fluorescence measurement after 24 and 48 hours. Transparent alpha-MEM medium was used to minimize background noise. We used the Varioskan LUX spectral scanning multimode reader to measure fluorescence levels. Excitation wavelength was 488nm, emission wavelength was 509nm, and bandwidth was 5nm. Result is shown in the figure below. Cultured cell control is taken from the supernatant of cells that have not been transfected with the internal control plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

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