Part:BBa_K3385066
T1G3R_ScbhB_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_cbhB 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 ΔglaA strain with the glaA expression cassette integrated (right) are albino compared to the dark spores of the ΔglaA 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 5762
Illegal EcoRI site found at 7190
Illegal XbaI site found at 1623
Illegal XbaI site found at 5793
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4024
Illegal PstI site found at 6600
Illegal PstI site found at 8334
Illegal PstI site found at 8935 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5762
Illegal EcoRI site found at 7190
Illegal NheI site found at 4901
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4024
Illegal PstI site found at 6600
Illegal PstI site found at 8334
Illegal PstI site found at 8935 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5762
Illegal EcoRI site found at 7190
Illegal BglII site found at 3879
Illegal BglII site found at 5401
Illegal BglII site found at 8432
Illegal BamHI site found at 1729
Illegal BamHI site found at 2157
Illegal BamHI site found at 4859
Illegal BamHI site found at 5661
Illegal BamHI site found at 5787
Illegal BamHI site found at 6338
Illegal XhoI site found at 548
Illegal XhoI site found at 735
Illegal XhoI site found at 963
Illegal XhoI site found at 6198 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5762
Illegal EcoRI site found at 7190
Illegal XbaI site found at 1623
Illegal XbaI site found at 5793
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4024
Illegal PstI site found at 6600
Illegal PstI site found at 8334
Illegal PstI site found at 8935 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5762
Illegal EcoRI site found at 7190
Illegal XbaI site found at 1623
Illegal XbaI site found at 5793
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4024
Illegal PstI site found at 6600
Illegal PstI site found at 8334
Illegal PstI site found at 8935
Illegal NgoMIV site found at 2566
Illegal NgoMIV site found at 5119
Illegal NgoMIV site found at 8322 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3407
Illegal BsaI site found at 3545
Illegal BsaI site found at 4234
Illegal BsaI.rc site found at 4727
Illegal SapI site found at 8823
Illegal SapI site found at 9132
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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