Team:TU Darmstadt/Composite Part
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 |
