Part:BBa_K1328003:Experience

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Lethbridge iGEM 2019 Improvement

Using the original design of BBa_K1328003 we have added a mRFP1 reporter on the N-terminus of the proinsulin and placed a furin cut site and linker in between. As well, we added additional furin cut sites and linkers to the original design. more info can be found on the parts pages BBa_K3237016,BBa_K3237017,BBa_K3237021 and BBa_K3237022. The parts BBa_K3237017 and BBa_K3237022 mentioned were also codon optimized for use in the microalgae C. reinhardtii . The following data is using part BBa_K3237016.

what we did

Figure 1: A 10% SDS-PAGE of the nickel affinity purification using the AKTA START system of the mRFP-proinsulin protein. Our protein is seen at between the 25kDa and 35kDa mark. This is lower than expected and is likely due to protease degredation.

Figure 2: A chromatogram of the proinsulin affinity purification. The blue line represents the A280 reads that correspond to our protein and the orange line corresponds to the amount of elution buffer (high imidazole content) used.

Furin Cleavage Assay

Although several attempts were made, we were unable to cut the proinsulin using the protease. 3.43, 2.58 and 2.38kDa bands were expected to be seen but as compared to the control, there was not change to the sample.

Figure 1: A 16.5% Tricine PAGE with mRFP1-Proinsulin and mRFP1-SCI57 treated with Furin Protease at 25 degrees Celsius for 16 hours. The gel was run at 100V for 10 minutes, then 180V for 60 minutes and stained with coomassie. Lane 1= 10-240kDA marker (Biobasic), lane 2= mRFP1-Proinsulin 10 units Furin, Lane 3= mRFP1-Proinsulin 20 units Furin, Lane 4= mRFP1-Proinsulin untreated, Lane 5= mRFP1-SCI57 10 units Furin, Lane 6= mRFP1-SCI57 20units Furin, Lane 7=mRFP1-SCi57 untreated.

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