Team:TU Darmstadt/Composite Part

TU Darmstadt

Parts



Listed in the following tables are all basic and composite parts that we created and submitted to the registry during our iGEM year. The parts we nominated for the best basic and composite part awards are marked with a ♥ in the tables. All parts we nominated for the best part collection are marked with a ♦. You can find the backbones used for cloning and characterization of the parts here .

Basic Parts


Best Basic Part ♥

For the best new basic part special award, we want to nominate our part BBa_K3187028, which encodes a Ca2+-independent hepta-mutant of the Sortase A enzyme, derived from Staphylococcus aureus, Sortase A7M. After successful expression and purification, we were able to test its functionality in a variety of different assays, e. g. FRET-based assays to understand the kinetics of the enzymes, screening for the best reaction conditions in the presence and absence of Ca2+ and even showed the possibility to fuse primary amines to LPETGG-tagged proteins. As we worked with the Ca2+-dependent Sortase A5M as well, we also compared the functionality of both variants. You can find all the results we were able to obtain on the corresponding registry page and our sortase wiki page.

Part (BBa_) Type Description Length in bp
BBa_K3187028 Basic (CDS) Sortase A7M (Ca2+-independent variant) 450
BBa_K3187016 Basic (CDS) Sortase A5M 450
BBa_K3187017 Basic (CDS) P22 Bacteriophage Coat Protein 1293
BBa_K3187018 Basic (Tag) GGGG-Tag for Sortase-mediated liagtion 12
BBa_K3187019 Basic (Tag) LPETGG-Tag for Sortase-mediated ligation 18
BBa_K3187020 Basic (CDS) TVMV protease cleavage site 18
BBa_K3187021 Basic (CDS) P22 Bacteriophage Scaffolding Protein 489
BBa_K3187022 Basic (CDS) Superfolder Green Fluorescence Protein 711
BBa_K3187023 Basic (CDS) TEV protease clevage site 21
BBa_K3187024 Basic (Tag) 6xHis-Tag or protein prufication + TGA Stop Codon 21
BBa_K3187025 Basic (Tag) Strep Tag II for protein purification 24
BBa_K3187026 Basic (CDS) mCherry Fluorescence Protein 705



Composite Parts


Best Composite Part ♥

For the best new composite part, we nominate the P22 coat protein with LPETGG-tag (BBa_K3187000). We chose this part because together with our nominee for the best new part it is the core of our project. The modification, and thus the modularity of our project, is based on sortase-mediated ligation of any protein of interest (POI) to the P22 Virus-like particle (VLP). For the covalent binding of the POI to the VLP the LPETGG-tag is necessary. Therefore, P22 CP with LPETGG-tag is our favorite part.
After successful expression and purification, we were able to show that together with the P22 scaffold protein (SP) VLPs could assemble and be purified over sucrose cushion and ultracentrifugation. Furthermore, we could show that the modification of our assembled VLPs utilizing Sortase A5M works. For this modification, we used GGGG-sfGFP as our POI.

Part (BBa_) Type Description Length in bp
BBa_K3187000 ♥♦ Composite P22 Bacteriophage Coat Protein with LPETGG Tag for Sortase-mediated Ligation 1650
BBa_K3187001 Composite P22 Bacteriophage Coat Protein 1667
BBa_K3187002 Composite P22 Bacteriophage Scaffolding Protein 782
BBa_K3187003 Composite Superfolder Green Fluorescence Protein x P22 Bacteriophage Scaffolding Protein 1547
BBa_K3187004 Composite TEV Cleavage Site x GGGG-Tag for Sortase-mediated Ligation X Superfolder Green Fluorescence Protein 1028
BBa_K3187005 Composite Sortase A7M (Ca2+-independent variant) 718
BBa_K3187006 Composite Sortase A5M 782
BBa_K3187008 Composite GGGG-Tag for Sortase-mediated Ligation x mCherry Fluorescence Protein 1074
BBa_K3187010 Composite TVMV Cleavage Site x GGGG-Tag for Sortase-mediated Ligation X mCherry Fluorescence Protein 1330
BBa_K3187011 Composite pT7 Superfolder Green Fluorescence Protein x pTet mCherry (Convergent) 2951
BBa_K3187012 Composite pTet sfGFP-P22 Bacteriophage Scaffolding Protein x Coat Protein with LPETGG Tag for Sortase-mediated Ligation pT7 (Convergent) 2951
BBa_K3187013 Composite P22 Coat and Scaffolding Protein x Coat Protein with LPETGG-tag for sortase-mediated ligation pT7 (Convergent) 3556
BBa_K3187014 Improved Plac-RBS-mCherry-double terminator (IPTG-inducible) with Translation Enhancing 5'UTR 1111
BBa_K3187015 Improved Constitutively expressed chromoprotein amilGFP with Translation Enhancing 5'-UTR 794
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