Revision as of 14:41, 19 October 2016

Signal peptide collection

Since signal peptides play an important role if you are woking on transmembrane constucts that are supposed to be secreted to the outer cell membrane we wanted to test some of the existing ones and submit new ones as well. 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.

BM40 signal peptide [http://parts.igem.org/Part:BBa_K2170214 BBa_K2170214]

Basement membrane 40 (BM40) signal peptide. This signal peptide showed the highest secretion of all peptids that we tested.

Ig Kappa signal peptide[http://parts.igem.org/Part:BBa_K2170215 BBa_BBa_K2170215]

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 with 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 with 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]