Construct design - Think before you print

While think before you print has been a popular slogan to oppose the excessive waste of paper in offices, it is also 100% applicable to our approach of bioprinting, as the depth of thought that goes into construct planning is generally proportional to the construct's functionality. In the following, we are going to explore the thought processes that went into the design of our receptor that mediates cross-linking of cells after printing, as well as the design of other functional parts involved in our project.

The receptor - linking by inking

The receptor that we created is able to cross-link cells after injection into streptavidin solution by presenting biotin moieties or streptavidin on the cell surface. This allows the almost irreversible interaction of the cell surface with streptavidin - either directly or via a coninjected biotinylated linker peptide - which binds to several cell surfaces at once, mediating cross-linking.

For the design of a receptor for intercellular cross-linking based on the previously presented concept, several elements for both function and detection as well as purification were considered and included into an expression construct. Of utmost importance is, of course, the correct anchoring of the receptor in the membrane, resulting in the correct localization of domains in either intercellular or extracellular space. For this purpose, a transmembrane domain of the human epidermal growth factor receptor (EGFR) was chosen. For correct trafficking of the receptor into the ER and, subsequently, its relocation on the cell surface, a properly functioning signal peptide had to be determined. Expression constructs containing three different and commonly used signal peptides from human trans- membrane proteins were tested. These include the signal peptide from human EGFR itself, the signal peptide of the human immunoglobulin kappa-chain as well as the osteonectin (also known as basement-membrane protein 40, short BM40) signal peptide. For quantification of the functionality of the signal peptide, secretion of a separately cloned pSB1C3 construct (being composed of only CMV promoter, signal peptide, nanoluciferase, Strep-tag II and a poly-A sequence) is being measured via a luciferase assay, measuring secretion into the cell medium and the final receptor construct adjusted accordingly. Moreover, the receptor contains three functional elements for its detection: An intracellular red fluorescent protein (mRuby) for detection of the receptor via flu- orescence microscopy, an extracellular nanoluciferase for detection via luciferase assays and an extracellular epitope domain for detection via A3C5-monoclonal antibodies (which may be coupled to secondary conjugates for detection via immunofluorescence or which may be used for immunoprecipitation). For purifi- cation of the receptor, it furthermore contains a C-terminal Strep-tag II. The functionality of the receptor is mediated by an extracellular biotin acceptor peptide (BAP) that is endogenously biotinylated by a coexpressed biotin ligase (BirA). The biotin ligase is hereby encoded by the same plasmid as the receptor, with an internal ribosome entry site - sequence (IRES) from the human eukaryotic translation initiation factor 4G1 gene (eIF4G1) allowing the polycistronic translation of two open reading frames (ORFs) from a single plasmid. BirA is targeted to the ER via an Igkappa signal peptide as well as an ER retention signal (also called KDEL sequence). Thus being permanently located in the ER, BirA is able to biotiny- late the BAP of translocating proteins upon their translocation, i.e. to the cell surface. The vector furthermore contains the poly-adenylation signal of human growth hormone (hGH) for functional polyadenylation of the transcribed mRNA. A schematic depiction of the vector is shown below. A description for the respective functional elements is given in the following sections.