Part:BBa_K2671000

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.

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

IMPROVEMENT REFERENCE: Linkoping_Sweden 2019

An improved version of this part can be seen here: BBa_K3182300 (Group: iGEM19_Linkoping_Sweden) Designed by: Oliver Hild Walett

Figure 4. Comparison of the old biobrick, BBa_K2671000 (light grey), and the new biobrick BBa_K3182300 (dark grey). Relative fluorescence divided by start OD₆₀₀ after 16 hours is shown. The error bars represent the mean ± SD from four technical replicates.

Expression

To evenly compare the parts, both biobricks were grown to a starting optical density at 600 nm (OD₆₀₀) of 0.4 ± 0.05. Both biobricks were inoculated in LB-Miller media containing 25 ug/mL chloramphenicol. After the cultures reached OD₆₀₀ 0.4, this part was induced using 0.5 mM IPTG and the old part was in induced with 1.5 mM L-arabinose. Aliquotes of the induced cultures were then placed in a 96-well plate and later analyzed with spectrometry. Fluorescence was then measured over 16 hours in 37 °C and excitation wavelength was 505 nm and emission wavelength was 525. The results from this experiment can be seen in Figure 4. As expected the pT7, the improved part, had a fluorescence which was more than twice than BBa_K2671000.

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