Design and Production of a Hydrogel Forming Polypeptide: Engaging High School Students in Protein Design

Bioinks are a class of hydrogel that have the potential to be the ink used in the creation of printed organs, connective tissue, and other important structures within the body. One class of material that may be a suitable bioink hydrogel is elastin-like polypeptides (ELPs), which are synthetic biopolymers inspired by the naturally existing connective tissue elastin. ELPs consist of a repeat pentapeptide sequence (GXGVP)n, where X is any of the 20 naturally existing amino acids other than proline. These biomolecules are capable of exhibiting environmental responsiveness when exposed to certain stimulus such as salt concentration, temperature, and pH, depending on their chain length and guest residue. This is dictated by the ELP inverse transition temperature (Tt) which affords it the property of being soluble below Tt and becoming insoluble above Tt. By changing the molecular structure, above Tt their association has the potential to result in the assembly of the ELPs into micelles and other microstructures.The creation of these proteins requires the use of molecular biology, namely producing recombinant DNA through protein engineering approaches. Molecular biology is a complex and rapidly expanding field, typically not addressed in full until the latter half of an undergraduate biology degree. However, the basic techniques can be implemented as straightforward protocols. This work establishes a robust set ofvmolecular biology protocols established with the goal of making this field more approachable to younger audiences, particularly to students in high school. Typically, safety concerns and a difficult barrier to entry prevented the inclusion of younger students in biotechnology, but this protocol establishes a safe and high yield system that gives students an introduction to this field while contributing to actual research projects.Of significance is the DNA assembly system used in this study, which uses a newer and more robust ligation technique for assembling large sequences of pentapeptides. The Gibson assembly system was exploited to expand the length of ELP molecules through subsequent rounds of cloning. Multiple round of cloning were performed on different ELP constructs, and the sequence data and system specifics are reported within. Novel triblock ELP polypeptides were created in this study, which are capable of physically crosslinking and forming hydrogels under specific conditions