Difference between revisions of "Part:BBa K3833000"

Revision as of 18:18, 21 October 2021

Streptococcal Collagen-Like Protein

Streptococcal Collagen-Like Protein (Scl2) was originally discovered in gram-positive S. pyogenes. It is a prokaryotic protein that can be used to mimic human collagen and has been shown to form stable triple helices[1].

Scl2 is easily expressed in E. coli and lacks the requirement for post-translational modifications; this makes it a useful alternative to recombinant human collagen expressed in eukaryotic organisms[1]. For these reasons, we chose Scl2 as the backbone for Collatrix — a collagen-mimetic gradient hydrogel for bone-soft tissue interfaces.

Scl2 can be modified to specific locations with common mammalian cell binding sites, such as fibronectin and integrin [3]. After is it produced, it can be cross-linked both chemically (with the use of glutaraldehyde) and with UV light in order to create hydrogels to serve as scaffolds for cell growth.

Figure 1. This shows a basic process diagram of how the cells will produce Scl2 in our system.

The sequence for this part originated from the S. pyogenes native protein sequence [4], and was codon-optimized [2] for expression in E. coli. Modeling The modeling for the production of Scl2 was based on the following equation:

Where sigma 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 μm/mL.

We modeled this production using differing Scl2 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.

Figure 2: Average half life

Figure 3: Maximal half life

Figure 4: Minimal half life

References:

[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.

Sequence and Features