Part:BBa_K3385064
T1G3R_SeglB_glaA
About the part: Insert of glaA expression cassette for A. niger. It is made to be integrated in albA, which will lead to white/yellow conidia upon correct integration in the genome. The insert is inserted in pSB1C3 via USER cloning to create the integration plasmid for A. niger. The signal peptide was changed to SigP_eglB using USER cloning and primer extension. The integration platform is made from part BBa_K3046030 pGIA2P1 designed by the DTU iGEM 2019 team.
Functionality: This signal peptide was investigated in Deeploc [1] to predict the localization of the protein which it is inserted upstream of.
Results:The efficiency of the signal peptide was tested through a starch/iodine plate assay, where no colouring shows breakdown of starch. However, this testing is not conclusive as other amylases may be overexpressed. Therefore, this part needs further testing.
Looking at the comparison pictures below, it is seen that the spores from the ÎglaA strain with the glaA expression cassette integrated (right) are albino compared to the dark spores of the ÎglaA strain (left). This shows success in the integration of the expression cassette, as albA is looped out.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5753
Illegal EcoRI site found at 7181
Illegal XbaI site found at 1623
Illegal XbaI site found at 5784
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4015
Illegal PstI site found at 6591
Illegal PstI site found at 8325
Illegal PstI site found at 8926 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5753
Illegal EcoRI site found at 7181
Illegal NheI site found at 4892
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4015
Illegal PstI site found at 6591
Illegal PstI site found at 8325
Illegal PstI site found at 8926 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5753
Illegal EcoRI site found at 7181
Illegal BglII site found at 3870
Illegal BglII site found at 5392
Illegal BglII site found at 8423
Illegal BamHI site found at 1729
Illegal BamHI site found at 2157
Illegal BamHI site found at 4850
Illegal BamHI site found at 5652
Illegal BamHI site found at 5778
Illegal BamHI site found at 6329
Illegal XhoI site found at 548
Illegal XhoI site found at 735
Illegal XhoI site found at 963
Illegal XhoI site found at 6189 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5753
Illegal EcoRI site found at 7181
Illegal XbaI site found at 1623
Illegal XbaI site found at 5784
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4015
Illegal PstI site found at 6591
Illegal PstI site found at 8325
Illegal PstI site found at 8926 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5753
Illegal EcoRI site found at 7181
Illegal XbaI site found at 1623
Illegal XbaI site found at 5784
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4015
Illegal PstI site found at 6591
Illegal PstI site found at 8325
Illegal PstI site found at 8926
Illegal NgoMIV site found at 2566
Illegal NgoMIV site found at 5110
Illegal NgoMIV site found at 8313 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3398
Illegal BsaI site found at 3536
Illegal BsaI site found at 4225
Illegal BsaI.rc site found at 4718
Illegal SapI site found at 8814
Illegal SapI site found at 9123
References:
[1] Jose Juan Almagro Armenteros, Casper Kaae Sønderby, Søren Kaae Sønderby, Henrik Nielsen, Ole Winther; DeepLoc: prediction of protein subcellular localization using deep learning, Bioinformatics, btx431
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