Team:LMU-TUM Munich/Design

Construct design

While think before you ink is a popular slogan by tattoo adversaries, it has also been 100% applicable to creating our approach of bioprinting. Careful planning is, of course, necessary to create a construct that fulfills its role in the most functional way possible. In the following, we are going to explore the thought processes that went into the design of the receptors we built to mediate cross-linking of cells for bioprinting, as well as the design of other functional parts involved in our project.

Design of synthetic receptors presenting biotin or biotin-binding proteins

Schematic depiction of the expression vector for the BAP-based receptor as well as the corresponding biotin ligase. Shown are the CMV promoter element, the signal peptide (depending on the construct being the EGFR signal peptide, the Igκ signal peptide or the BM40 signal peptide, the latter two furthermore possessing an elongated 5’ UTR), the biotin acceptor peptide, a nanoluciferase, an epitope tag for A3C5 antibodies, the EGFR N-terminal transmembrane α-helix, mRuby, a Strep-tag II, an IRES from eIF4G1 for polycistronic transcription, BirA including a Igκ signal peptide as well as an ER retention signal sequence, and the hGH polyadenylation signal sequence.

The receptor we created is able to cross-link cells after injection into streptavidin solution by allowing the connection of several biotinylated cell surfaces through the tetravalent binder streptavidin. This reaction being very fast and highly affine - with a K_D of 10^-15 M^-1 - allows precise positioning and cross-linking of cells for the creation of cellular matrices by bioprinting. For this project, we have created receptors for cross-linking of both human as well as bacterial cells.

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