Part:BBa_K4606011

Vector- Luciferase-SMAD4(WT)-AmpR

Description

For detailed design information, you can look up the registry of BBa_K4606007, BBa_K4606002 and BBa_K4606003.

In this part, we integrated the fragment sequence of the SMAD4(WT) gene in front of the luciferase gene and loaded them together into the plasmid vector.(Figure 1.)

Figure 1. Expression map of the composite plasmids of SMAD4-WT.

This segment of SMAD4(WT) will then be transcribed and expressed together with the luciferase gene in cells. If SMAD4(WT) gene is inhibited by miRNA, its downstream luciferase will not be expressed normally. Therefore, we could analyze whether SMAD4(WT) gene was inhibited by examining the luciferase activity.

Due to the addition of the AmpR gene to the plasmid, we could easily confirm whether the recombination was successful or not, as only the recombinants can grow into colonies on the medium supplemented with Ampicillin.

We identified the plasmid construction by restrictive endonuclease digestion and agarose gel electrophoresis (Figure 2).

Figure 2. Verification of the recombinant plasmid of SMAD4-WT.
1#: Blank control (ddH2O).
2#: Negative control (empty vector without target gene).
3#: Positive control (GAPDH gene was used as a template).
4#: DNA marker.
5-9#: The recombinant plasmid after transformation.

We transfected the recombinant plasmid of SMAD4-WT into chondrocytes. After 24 h, luciferase activity could be detected in chondrocytes. Interestingly, concurrent transfection of miR-3074-5p composite plasmids significantly reduced luciferase activity (Figure 3).

Figure 3. Relative luciferase activity in chondrocytes after transfection the recombinant plasmid of SMAD4-WT.

Design

This device is used to transfer the coding gene of SMAD4 (WT) fragment and luciferase into chondrocytes. Due to the addition of the AmpR gene to the plasmid, we could easily confirm whether the recombination was successful or not, as only the recombinants can grow into colonies on the medium supplemented with Ampicillin.

Source

Plasmids were obtained from Escherichia coli.

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]