Demonstrate
Our system relies on recombinant E. coli to produce Scl2, a collagen-like protein. Once this peptide is produced and purified, it can be cross-linked to form a polymer gel. The gel is formed with a gradient of integrin and fibronectin-binding sites using a gradient maker designed by our team. This gradient of binding sites will allow fibroblasts and other cells that make up epithelial and connective tissue to bind and grow into the gel.
Figure 1 shows a basic process diagram of how the cells will produce collagen in our system.
The modeling for the production of Scl2 was based on the following equation:
Where σ is the basal production rate of Scl2 based on our constitutive promoter, x is the number of E. coli, R is the degradation rate of Scl2, and P is the concentration of Scl2 in um/mL.
Modeling
We modeled this production using differing Scl_2 half-lives. We used three different half-lives based on degradation rates found during the literature review. Figure 2 plots an average half-life, while figures 3 and 4 plot maximal and minimal protein degradation respectively. While we expect the plot in figure 2 to be most realistic, we modeled three different situations to make sure that the Scl2 would be at a sufficient concentration to cross-link in both minimal and maximal degradation conditions. 
Lab Work
Due to time constraints and COVID-related shipping delays, the full extent of our team’s lab work was unable to be completed. Synthesized gene fragments for our DNA segments of interest were obtained from Invitrogen’s GeneArt Gene Synthesis service. Parts BBa_K3833005, BBa_K3833006, and BBa_K3833007 were synthesized using this service. PCR primers were ordered from Integrated DNA Technologie as custom DNA oligos. The pET15-MHL plasmid backbone containing a polyhistidine tag was obtained from addgeneFirst, our team used PCR to amplify our composite Scl2 with fibronectin binding domain gBlock with our custom primers. After amplification, a gel electrophoresis was run on the PCR product using 1Kb Plus DNA Ladder. A significant band was obtained for the PCR, around 1,400bp (Picture 1). Because we predicted this composite part to be 1,394 bp, it was determined that amplification of our Scl2 with fibronectin binding domain successfully occurred.
Picture 1: Gel Electrophoresis (1Kb Plus Ladder) of Part:BBa_K3833007 — Scl2 with Fibronectin Binding Domain PCR Product
We then ran a wide comb gel electrophoresis on the remaining PCR product and recovered our amplified DNA using the Monarch DNA Gel Extraction Kit protocol. Then, the concentration of the extraction protocol DNA eluate was found using absorbance measurements at 260nm by means of nanodrop (Table 1).
Cell Culture
Background
As another measure of testing, our hydrogels were seeded with Mouse Fibroblast L929 cells. This cell line expresses the 𝛼1 Integrin subunit protein which allows it to bind to Integrin binding sites. Collatrix hydrogels are made up of a gradient of fibronectin and integrin-binding sites, so our goal was to determine whether mammalian cells would grow and survive within the gel.As a proof of concept, cells were seeded onto gelatin and rat tail collagen I hydrogels in order to test the potential cytotoxicity of the materials themselves and the potential drawbacks of the cross-linking mechanisms used.
Gelatin Hydrogels
Approximately 1 uL of Mouse Fibroblast L929 were seeded on top of the hydrogels. In addition to being seeded on top of the gels, the tip of the micropipette was inserted into the gels in order to place cells inside the gel matrix.After allowing the cells to grow for approximately 24 hours, the cells were stained with 1 ug/mL Hoechst 33342 dye to visualize living cell nuclei (blue) and 1 ug/mL propidium iodide to visualize dead cells (red.) The gels were imaged using an epi-fluorescent microscope on 20x and 40x objectives. Many of the dead cells seemed to have detached from the hydrogels, leading to only a few remaining living cells visible in the images (Fig. 1).
Figure 1 Cells cultured in the presence of gelatin hydrogel crosslinked with glutaraldehyde imaged at 20x (A) and 40x (B) magnification
Collagen Hydrogels
The collagen hydrogels were seeded, stained, and imaged using the same protocol as described for the gelatin hydrogels. There were 5 different collagen gels tested, one each of collagen and collagen with nHap particles without any crosslinking agent, one each of collagen and collagen with nHap particles crosslinked with glutaraldehyde, and one collagen gel crosslinked with UV light manufactured as described in Hydrogel Creation Process.When the same protocol used on the gelatin hydrogels was performed on these gels, results differed based on gel composition and cross-linking method. The gels without any crosslinking method dissolved in the media overnight and were not viable to be imaged. The gels crosslinked with glutaraldehyde both caused the media to turn yellow overnight, indicating issues with cell viability. The pure collagen version had a few viable cells, but a majority of the cells died overnight and detached. However, some of the dead cells remained attached and can be seen next to their living counterparts (Fig. 2 B, B’) The collagen and nHap hydrogels did not yield any viable cells, so no images were abel to be taken of proliferated cells.The UV gels were the most successful, and displayed several viable attached cells after 24 hours (Fig. 2C, D)
Figure 2 Cells cultured in the presence of collagen hydrogels crosslinked with glutaraldehyde (A, B, B’) and UV light (C, D) imaged at 20x (A, C) and 40x (B, B’, D) magnification.
From these results, we gather that Collatrix should be cross-linked with UV light only in order to prevent potential cytotoxicity from cross-linking agents like glutaraldehyde. We also know that the gels need to be cross-linked using an external method (ex. light) in order to keep the hydrogels cross-linked for extended periods of time: the cross-linking that collagen protein does on its own is not sufficient in a highly aqueous environment.
Resources
- [1] An, B., Kaplan, D. L., & Brodsky, B. (2014). Engineered recombinant bacterial collagen as an alternative collagen-based biomaterial for tissue engineering. Frontiers in Chemistry, 2, 40. https://doi.org/10.3389/fchem.2014.00040
- [2] Codon Optimization. (n.d.). VectorBuilder. Retrieved from https://en.vectorbuilder.com/tool/codon-optimization.html.
- [3] Parenteau-Bareil, R., Gauvin, R., & Berthod, F. (2010). Collagen-Based Biomaterials for Tissue Engineering Applications. Materials, 3(3), 1863–1887. https://doi.org/10.3390/ma3031863
- [4] UniProtKB - Q8RLX7. (n.d). UniProt. Retrieved from https://www.uniprot.org/uniprot/Q8RLX7.
- [5] Bionumers. Typical Protein Half-Life. Retrieved from https://bionumbers.hms.harvard.edu/bionumber.aspx?s=n&v=2&id=111930
- [6] Maurizi, M.R (1992). Proteases and Protein Degradation in Escherichia coli. Laboratory of Cell Biology, National Cancer Institute, Bethesda, 48. Retrieved from https://link.springer.com/content/pdf/10.1007/BF01923511.pdf.
- [7] Peng, Yong Y., Yoshizumi, Ayumi, Danon, Stephen J., Glattauer, Veronica, Prokopenko, Veronica, Mirochnitchenko, Oleg, Yu, Zhuoxin, Inouye, Masayori, Werkmeister Jerome A.,Brodsky, Barbara, Ramshawa, John A.M. A Streptococcus pyogenes derived collagen-like protein as a non-cytotoxic and non-immunogenic cross-linkable biomaterial. Biomaterials 31(10), 2009. https://doi.org/10.1016/j.biomaterials.2009.12.040.
- [8] Pletnev P, Osterman I, Sergiev P, Bogdanov A, Dontsova O. Survival guide: Escherichia coli in the stationary phase. Acta Naturae. 2015;7(4):22-33.