Part:BBa_K3385063
T1G3R_SeglA_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_eglA 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 5747
Illegal EcoRI site found at 7175
Illegal XbaI site found at 1623
Illegal XbaI site found at 5778
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4009
Illegal PstI site found at 6585
Illegal PstI site found at 8319
Illegal PstI site found at 8920 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5747
Illegal EcoRI site found at 7175
Illegal NheI site found at 4886
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4009
Illegal PstI site found at 6585
Illegal PstI site found at 8319
Illegal PstI site found at 8920 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5747
Illegal EcoRI site found at 7175
Illegal BglII site found at 3864
Illegal BglII site found at 5386
Illegal BglII site found at 8417
Illegal BamHI site found at 1729
Illegal BamHI site found at 2157
Illegal BamHI site found at 4844
Illegal BamHI site found at 5646
Illegal BamHI site found at 5772
Illegal BamHI site found at 6323
Illegal XhoI site found at 548
Illegal XhoI site found at 735
Illegal XhoI site found at 963
Illegal XhoI site found at 6183 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5747
Illegal EcoRI site found at 7175
Illegal XbaI site found at 1623
Illegal XbaI site found at 5778
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4009
Illegal PstI site found at 6585
Illegal PstI site found at 8319
Illegal PstI site found at 8920 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5747
Illegal EcoRI site found at 7175
Illegal XbaI site found at 1623
Illegal XbaI site found at 5778
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4009
Illegal PstI site found at 6585
Illegal PstI site found at 8319
Illegal PstI site found at 8920
Illegal NgoMIV site found at 2566
Illegal NgoMIV site found at 5104
Illegal NgoMIV site found at 8307 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3392
Illegal BsaI site found at 3530
Illegal BsaI site found at 4219
Illegal BsaI.rc site found at 4712
Illegal SapI site found at 8808
Illegal SapI site found at 9117
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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