Part:BBa_K1655001

This GFP can be fused into any protein's aminoterminal end with the BioBrick enzyme assembly method.

An extra nucelotide has been added prior to this GFP brick's suffix to maintain the reading frame after fusion.

Usage and Biology

This part sequence is derived from https://parts.igem.org/Part:BBa_I13504. The part has been PCRed from BBa_I13504, and the reverse primer has added an extra guanine nucleotide prior to the birck's suffix. This will allow for easy aminoterminal GFP fusion with the biobrick enzyme assembly.

Fusable GFP / BBa_K1655001

Figure 1. GFP Brick

We built a GFP biobrick which can be fused to any protein's aminoterminal end with the standard BioBrick assembly enzymes. We have added an extra nucleotide prior to the brick's suffix to maintain the reading frame after fusion, which is typically lost when the restriction enzyme assembly is used.

Validation: Our GFP brick has been fully sequenced, and the sequencing results were as expected. See figure 2. for the sequencing results. We have also been able to express the GFP after fusing it with an amphiphilic brick. This construct functioned under BBa_K608003, a strong constitutive promoter and a medium RBS. HS Slovenia Team also helped us validate this brick. They gained positive results of their construct with the GFP through colony PCR and analytical restrictions, but were unable to detect the fluerescence under UV light or functionality of the fused protein. Figure 3 shows a positive result of colony PCR. The GFP has indeed been fused with another protein, CtfB, with the biobrick enzyme assembly. We were however able to show that the GFP is functional after fusion. See figure 4. for microscopic pictures.

We restricted the GFP brick with XbaI & PstI to show that the insert in the brick was of the correct size. DNA from the colony which produced the band seen in Figure 5 in well 4 was sent to the registry.

Click here to download the full sequence of our Fusable GFP in pSB1C3 backbone.

Click the images to enlarge them

Figure 2. GFP brick's sequencing results

Figure 3. Slovenia's colony PCR showing that GFP is fused with CtfB

Figure 4. Microscopic images of GFP fusion

Figure 5. GFP brick restricted to show the right-sized insert.

This brick is a twin to three different bricks. We checked the BioBrick seeker for any similar bricks before we started with our project, and the twin for this part did not come up. We only realized that a brick like this had already been created after we submitted our parts, which was about a week prior to the wiki freeze.

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]

  Illegal BsaI.rc site found at 644