Propane 1 / BBa_K1655000

Our Propane 1 includes 3 of the 10 required genes to produce propane in E. coli. The plasmid has been assembled from IDT's gBlocks with NEBuilder assembly, similar to Gibson Assembly.

Validation: We restricted our Propane 1 and ran the insert on an agarose gel. From the picture we can tell that the insert is the correct size. The result can be seen in Figure 2.
Additionally, we did a colony PCR with VR and our primer P001. The VR primer attaches to our plasmid's backbone while P001 anneals with the very beginning of our construct. With this colony PCR we were able to show that the insert is present and it is indeed in the pSB1C3 backbone. See figure 3 for results, where the product in wells 1, and 5-10 is of the right size.

The colony which produced the product seen in figure 3, well 10 was sent to the registry under the name BBa_K1655000.

Click here to download the full sequence of Propane 1 in pSB1C3 backbone.

Click the images to enlarge them.

Fusable GFP / BBa_K1655001

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 5. 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 6 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 7. 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 8 in well 4 was sent to the registry under the name BBa_K1655001.

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

Click the images to enlarge them

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.

Amphiphilic / BBa_K1655002

We submitted a sole amphiphilic brick, containing only the coding region of the ampihphilic protein. This brick can be used to produce intracellular micelles or vesicles under any chosen expression system. Our amphiphilic protein is constructed so that the hydrophilic part is translated first. According to Huber et al. this allows for the formation on of intracellular micelles and vesicles. If the hydrophobic domain was to be translated first, the amphiphils would only form micelles.

Validation: Our amphiphilic brick's sequencing results were unclear, as the brick is mainly built of short repeats. Microscope pictures!

The colony which produced the product seen in figure 10, well 10 was sent to the registry under the name BBa_K1655002.

Click here to download the full sequence of the Amphiphilic Brick in pSB1C3 backbone.