Part:BBa_K3385065
T1G3R_ScbhA_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_ScbhA 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 5750
Illegal EcoRI site found at 7178
Illegal XbaI site found at 1623
Illegal XbaI site found at 5781
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4012
Illegal PstI site found at 6588
Illegal PstI site found at 8322
Illegal PstI site found at 8923 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5750
Illegal EcoRI site found at 7178
Illegal NheI site found at 4889
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4012
Illegal PstI site found at 6588
Illegal PstI site found at 8322
Illegal PstI site found at 8923 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5750
Illegal EcoRI site found at 7178
Illegal BglII site found at 3867
Illegal BglII site found at 5389
Illegal BglII site found at 8420
Illegal BamHI site found at 1729
Illegal BamHI site found at 2157
Illegal BamHI site found at 4847
Illegal BamHI site found at 5649
Illegal BamHI site found at 5775
Illegal BamHI site found at 6326
Illegal XhoI site found at 548
Illegal XhoI site found at 735
Illegal XhoI site found at 963
Illegal XhoI site found at 6186 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5750
Illegal EcoRI site found at 7178
Illegal XbaI site found at 1623
Illegal XbaI site found at 5781
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4012
Illegal PstI site found at 6588
Illegal PstI site found at 8322
Illegal PstI site found at 8923 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 1858
Illegal EcoRI site found at 5750
Illegal EcoRI site found at 7178
Illegal XbaI site found at 1623
Illegal XbaI site found at 5781
Illegal SpeI site found at 1097
Illegal PstI site found at 893
Illegal PstI site found at 4012
Illegal PstI site found at 6588
Illegal PstI site found at 8322
Illegal PstI site found at 8923
Illegal NgoMIV site found at 2566
Illegal NgoMIV site found at 5107
Illegal NgoMIV site found at 8310 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3395
Illegal BsaI site found at 3533
Illegal BsaI site found at 4222
Illegal BsaI.rc site found at 4715
Illegal SapI site found at 8811
Illegal SapI site found at 9120
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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