Part:BBa_K4585012

pcDNA3.1(+)-3XHA-GAL4-VP64-NLS

The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid, which could express GAL4-VP64, was used for Luciferase detection experiment. GAL4 is a protein that can find and bind UAS (upstream activation sequence). VP64 is a transcription factor that, when used in combination with GAL4, can activate UAS and initiate the expression of downstream genes.

pcDNA3.1(+)-3×HA-GAL4-VP64-NLS

The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid was obtained through homologous recombination of the VP64 homologous recombination insert (BBa_K4585002) with pcDNA3.1(+)-3×HA-GAL4-VP64-NLS linearized vector (BBa_K4585006). The homologous recombination plasmid product was identified as the target product by sequencing and enzyme cutting and agarose gel electrophoresis.

1 Pattern Diagram

Fig.1 The model diagram of pcDNA3.1(+)-3×HA-GAL4-VP64-NLS

2.1 Method

The pcDNA3.1(+)-3×HA-GAL4-VP64-NLS plasmid could express GAL4-VP64, thereby activating 9×UAS, which could activate the expression of its downstream gene, GAL4-KRAB or Luciferase.

2.2 Results

Insulin can be processed and secreted outside the cell in 293T cells.

Fig.2 Standard curve of absorbance and insulin concentration

Fig.3 Insulin concentration in supernatant after blue light irradiation and dark treatment respectively

Fig.4 Insulin concentration in supernatant (excluding insulin in medium) after blue light irradiation and dark treatment respectively

3.Caution

Insulin is not suitable for long time preservation under the best culture temperature of cells (37℃). In the mean time, cells would also consume some of the expressed insulin. So the best tst time for Insulin is approximately 24 hours after transfection, it is relatively shorter than the test interval of LUC.

Reference：

[1]Mingqi Xie, Haifeng Ye, Hui Wang.β-cell-mimetic designer cells provide closed-loop glycemic control[J].Science.2016 Dec 9;354(6317):1296-1301.

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
  COMPATIBLE WITH RFC[1000]