Team:LMU-TUM Munich/Part Collection

BioBrick part collections

HypoxiaSensor collection

Since the growth of tissue above a certain diameter leads to hypoxia of the cells in the centre we wanted to adress this problem with the constructs below.

[http://parts.igem.org/Part:BBa_K2170030 BBa_K2170030]

Vascularization inducer

Hypoxia response elements downstream of a minipromotor that regulates the expression of vascular endothelial growth factor (VEGF) that induces vascularisation of tissue by endothelial cells.

[http://parts.igem.org/Part:BBa_K2170031 BBa_K2170031]

VEGF

Constitutive

[http://parts.igem.org/Part:BBa_K2170040 BBa_K2170040]

Hypoxia sensor

[http://parts.igem.org/Part:BBa_K2170041 BBa_K2170041]-[http://parts.igem.org/Part:BBa_K2170044 BBa_K2170044]

Testing device for Hypoxia sensor

Nanoluciferase downstream of a minipromoto which is regulated by a different number of upstream hypoxia response elements, which induce the expression under hypoxic conditions. The capacity of the response elements to react to hypoxic conditions confirmed by NanoGlo-Assay.

Signal peptide collection

To achieve the best possible secretion of our eucaryotic receptors we tested three different signal peptides for their secretion efficiency. The expression of all testing devices was regulatd by a cyto megalo virus (CMV) promotor. The secretion efficiency was tested by NanoGlo-Assay.

[http://parts.igem.org/Part:BBa_K2170010 BBa_K2170010]

BM40 secretory Nanoluciferase

Fusion protein with Basement membrane 40 (BM40) signal peptide.

[http://parts.igem.org/Part:BBa_K2170011 BBa_K2170011]

EGFR secretory Nanoluciferase

Fusion protein with epidermal growth factor receptor (EGFR) signal peptide.

[http://parts.igem.org/Part:BBa_K2170012 BBa_K2170012]

IgKappa secretory Nanoluciferase

Fusion protein with immunoglobulin Kappa (IgKappa) signal peptide.

[http://parts.igem.org/Part:BBa_K2170013 BBa_K2170013]

non-secretory Nanoluciferase

The non-secretory Nanoluciferase testing device which does not contain a signal peptide was used to obtain a baseline signal.

Eucaryotic receptor collection

All eucaryotic receptors that we designed share a similar structure and were developed for use in eucaryotic cells. Due to the intracellular mRuby3 the general expression and location of the receptor can be detected by fluorescence signal. Additionaly we integrated an antibody binding site (A3C5 Tag) and a Strep Tag for better characterisation.

[http://parts.igem.org/Part:BBa_K2170000 BBa_K2170000]

Bap-NanoLuc receptor construct

Fusion protein with a Biotin acceptor peptide (BAP) for intracellular biotinylation by biotin ligase BirA from E. coli. The Biobrick contains an internal ribosomal binding site in front of the BirA so that it, although it is part of the same mRNA, is translated seperately from the rest of the transcript. The fusion protein is directed to the cellmembrane where it presents the biotinylated BAP on the surface. At the C terminal end of the BAP a Nanoluciferase was introduced so that the correct location of the receptor could be tested by NanoGlo-Assay.

[http://parts.igem.org/Part:BBa_K2170001 BBa_K2170001]

Biotin binding receptor + eMa

Fusion protein with an enhanced monomeric avidin (eMA). This monomeric avidin like protein is presented on the cellsurface, where it can bind extracellular biotin.

[http://parts.igem.org/Part:BBa_K2170002 BBa_K2170002]

Biotin binding receptor + scAvidin

Fusion protein with an single chain avidin (scAvidin). This cyclic permutated avidin is presented on the cellsurface, where it can bind extracellular biotin.

Procaryotic receptor collection

All procaryotic receptors we designed share a similar structure. The expression is regulated by a TetR-TetO constuct which can be induced by Tetracycline. The secretion signal is an Outer membrane protein A (OmpA) signal peptide. The EspP autotransporter leads to a presentation of the receptor on the cell surface. The intruduction of the antibody binding site (A3C5 Tag) can be used as indicator for the right localisation of the receptor.

[http://parts.igem.org/Part:BBa_K2170051 BBa_K2170051]

BAP-Nanoluc receptor construct

[http://parts.igem.org/Part:BBa_K2170050 BBa_K2170050]

Biotin binding receptor with eMA

[http://parts.igem.org/Part:BBa_K2170052 BBa_K2170052]

Biotin binding receptor with scAvidin

[http://parts.igem.org/Part:BBa_K2170053 BBa_K2170053]

Secretory Nanoluciferase

Optogenetic Ca2+ signaling device

Kill-switch collection

[http://parts.igem.org/Part:BBa_K2170060 BBa_K2170060]

Kill-switch with polyA

[http://parts.igem.org/Part:BBa_K2170150 BBa_K2170150]

Kill-switch without polyA

<groupparts>iGEM2016 LMU-TUM_Munich</groupparts>