Difference between revisions of "Part:BBa K2671000"

Revision as of 21:35, 17 October 2018

AraC-pBAD-mNG

iGEM17_Linkoping_Sweden part BBa_K2474000 modified. A stop codon was mutated behind mNeonGreen (mNG). The original part was mNG fused to Aβ1-42, where the fusion protein had its stop codon after Aβ1-42. Another mutation was made in the GS-linker sequence where a SpeI-site was added and the sequence for Aβ1-42 was cleaved off. With these improvements mNG can now be used as a reporter in a fully functional operon with the pBAD-AraC inducible system. It also compares the difficulty an aggregation-prone fusion protein to a non fused version.

Usage and Biology

Results

The results for our improved part can be seen below. The reason we chose to improve this part was not only to create a functional reporter system, but also to illustrate the loss of exchange with a aggregation prone fusion tag. From Figure 1. you can clearly see that the version without Aβ1-42 folds better and thus gives a much higher fluorescence, while the fusion version is having trouble. The two parts was grown in LB-medium, containing 0.25 mg/ml L-arabinose and 25 ug/ml chloramphenicol over 16 hours. They were then lysed through the "freeze-thaw" method and centrifuged. The supernatant was saved and placed on an UV table as seen in Figure 1.

Figure 1. From left to right: Reference, mutated version (this biobrick) and the unmutated version (BBa_K2474000).

Sequencing

As seen in Figure 1. The improved version has a much stronger color, which indicates that the Aß1-42 fusion was successfully terminated. And as seen in Figure 2 and 3. The sequencing results confirms this. File:T--Linkoping Sweden--BBa K2671000seq.zip Sequencing data files. The analyzed data in Figure 2 and 3. is from these files. These contain full information on the sequencing including a chromatogram.

Figure 3. Comparing the sequencing results of the mutated version (this biobrick) and the template (BBa_K2474000). Showing a successful addition of an stop codon. Comparing the length of the sequence, both done with the VR primer, a loss of ~170 bases. This confirms a successful removal of the Aβ1-42-tail.

Figure 2. Sequencing results (see attached files for an chromatogram) analyzed in benchling, annotations added for clarity. The sequencing data attached confirms a successful removal of Aβ1-42 and addition of a stop codon. Sequencing was done with the VR primer.

IMPROVEMENT REFERENCE: Linkoping_Sweden 2018

This part is a modified version of BBa_K2474000 (Group: iGEM17_Linkoping_Sweden), Designed by: J. Baggman, J. Bergqvist, H. Karlsson, L. Karlsson, J. Larsson, M. Lindberg, M. Nilsson, M. Peterson, O. Reinhed Gustafsson, S. Stridh Karppinen & J. Ybrahim

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1205
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1267
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961

@@ Line 8: / Line 8: @@
 <h2>Results</h2>
-The results for our improved part can be seen in below. The reason we choose to improve this part was not only to create a functional reporter system, but also to illustrate the loss of exchange with a aggregation prone fusion tag. From fig 1. you can clearly see that the version without amyloid-beta 1-42 folds better and thus gives a much higher fluorescence, while the fusion version is having trouble. The two parts was grown in LB-medium, 0.25 mg/ml L-arabinose and 25 ug/ml chloramphenicol over 16 hours. They were then lysed through the "freeze-thaw" method and centrifuged. The supernatant was saved and placed on an UV table as seen in fig 1.
+The results for our improved part can be seen below. The reason we chose to improve this part was not only to create a functional reporter system, but also to illustrate the loss of exchange with a aggregation prone fusion tag. From Figure 1. you can clearly see that the version without Aβ1-42 folds better and thus gives a much higher fluorescence, while the fusion version is having trouble. The two parts was grown in LB-medium, containing 0.25 mg/ml L-arabinose and 25 ug/ml chloramphenicol over 16 hours. They were then lysed through the "freeze-thaw" method and centrifuged. The supernatant was saved and placed on an UV table as seen in Figure 1.
 [[File:T--Linkoping_Sweden--improved.jpeg|450px|thumb|left|Figure 1. From left to right: Reference, mutated version (this biobrick) and the unmutated version (BBa_K2474000). ]]
@@ Line 37: / Line 37: @@
 <br>
 <h2>Sequencing</h2>
-As seen in fig 1. the improved version has a much stronger color, which indicates that the Aß1-42 fusion was successfully terminated. And as seen in fig 2, 3. the sequencing results confirms this.
+As seen in Figure 1. The improved version has a much stronger color, which indicates that the Aß1-42 fusion was successfully terminated. And as seen in Figure 2 and 3. The sequencing results confirms this.
 [[File:T--Linkoping Sweden--BBa K2671000seq.zip|200px|thumb|right|]]
-Sequencing data files. The analyzed data in figure 2 and 3. is from these files. These contain full information on the sequencing including a chromatogram.
+Sequencing data files. The analyzed data in Figure 2 and 3. is from these files. These contain full information on the sequencing including a chromatogram.
 [[File:T--Linkoping_Sweden--chromatogram.png|450px|thumb|center| Figure 3. Comparing the sequencing results of the mutated version (this biobrick) and the template (BBa_K2474000). Showing a successful addition of an stop codon. Comparing the length of the sequence, both done with the VR primer, a loss of ~170 bases. This confirms a successful removal of the Aβ1-42-tail. ]]