Difference between revisions of "Part:BBa K1655002"

Latest revision as of 07:55, 18 September 2015

Amphiphilic proteins can be used to form intracellular micelles or vesicles.

This is the coding sequence for an amphiphilic protein. The protein has both a hydrophilic and a hydrophobic domain, each about 300bps in length. In this brick, the hydrophilic domain is translated first. According to Huber et al. this allows for the formation of intracellular vesicles rather than micelles. The coding sequence is derived from this article.

Usage and Biology

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 of this insert in the pSB1C3 backbone is correct. See figure 2 for a gel electrophoresis image. DNA from the colony which produced the product seen in figure 10, 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 3 where the last two wells before the ladder show a correct plasmid size. Although the sequencing results are unclear, the correct sized mRNA seemed to be produced. This lead us to believe, that the Amphiphilic protein was indeed transcribed and possibly translated as well. We thought that 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 was 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 2). 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.

Click the images to enlarge them

**Figure 3.** GFP-Amphiphilic fusion on gel

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 616
23
INCOMPATIBLE WITH RFC[23]
Unknown
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 13
1000
COMPATIBLE WITH RFC[1000]

@@ Line 10: / Line 10: @@
 <!-- Add more about the biology of this part here-->
 ===Usage and Biology===
-<html><b>Validation:</b> 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 of this insert in the pSB1C3 backbone is correct. See figure 2 for a gel electrophoresis image. DNA from the colony which produced the product seen in figure 10, 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 3 where the last two wells before the ladder show a correct plasmid size. 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.<br/><br/>
+<html><b>Validation:</b> 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 of this insert in the pSB1C3 backbone is correct. See figure 2 for a gel electrophoresis image. DNA from the colony which produced the product seen in figure 10, 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 3 where the last two wells before the ladder show a correct plasmid size. Although the sequencing results are unclear, the correct sized mRNA seemed to be produced. This lead us to believe, that the Amphiphilic protein was indeed transcribed and possibly translated as well. We thought that 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 was 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.<br/><br/>
 <span style="color:red"><b>Note!</b></span> 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 2). 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 <a href="https://parts.igem.org/Part:BBa_K592009">blue chromoprotein</a>.<br/>