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 plasmid's size is correct. 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.
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
Figure 5. GFP brick's sequencing results
Figure 6. Slovenia's colony PCR showing that GFP is fused with CtfB
Figure 7. Microscopic images of GFP fusion
Figure 8. 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.
Amphiphilic / BBa_K1655002
Figure 9. Amphiphilic Brick
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. We have however been able to show that the size on this insert in the pSB1C3 backbone is correct. See figure 10 for a gel electrophoresis image. The DNA from the colony which produced the product seen in well 2 was sent to the registry. We were also able to show, that once this Amphiphilic brick is fused to a GFP, the GFP is expressed and again, the construct size is correct. See figure 11. Although the sequencing results are unclear, there seemed to be no premature stop codons in the sequence. This leads us to believe, that the Amphiphilic protein was indeed translated. We weren't able to detect the micelles and vesicles because of low accuracy of the microscopic images or because our expression level was too high. Our promoter is 10 times more efficient than the one that has been previously used to produce micelles and vesicles with this amphiphilic protein. The high concentration of amphiphils is likely to interfere with the micelle and vesicle formation.
Note! The submitted BioBrick does most likely not contain the correct sequence. Restriction analysis lead us to initially believe we had the correct insert, as the insert size in pSB1C3 backbone matched that of the correct sequence (figure 10). However, as we proceeded to the sequencing results of the then already submitted brick a day before the wiki freeze, we found out that the sequence was not that of the amphiphilic protein, but instead that of the blue chromoprotein.
