Part:BBa_K1583102:Design

pRha + CsgA + Hydroxyapatite-affinity tag

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

In our research about the dental surface we found that the external part that protects the inner tooth fragments is the enamel. Hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), also known as bone mineral, is a mineral compound that represents the major inorganic part of the dental enamel and dentin (Staines,M. et al, 1981). Thus, we looked forward to a way to make our biofilm attach better into a hydroxyapatite-covered surface.

In the end, we found that our homemade biofilm could be improved with surface-specificity just by adding a particular tag attached to our amyloid protein, CsgA (Roy,M. et al, 2008). This tag, successfully added to our csgA construct and cloned in our vehicle strain, does successfully make biofilms when induced with rhamnose. However, we thought that the best way to prove that it can resemble a real mouth-attached biofilm is testing it with teeth! (Figure 1)

Figure 1: Teeth from a cow (Bos taurus) used in the experiment for testing the hydroxyapatite peptide tag

Source

This part was synthesized. The rhamnose promoter was used by the iGEM 2014 TU Delft team and originally added by the iGEM12 Paris Bettencourt team. RBS and CsgA originate from (E. coli K-12 MG1655). The nucleotides encoding for the second and third amino acid of the CsgA gene were changed to optimize synthesis success. (silent mutations).

References

Roy, M. D., Stanley, S. K., Amis, E. J., & Becker, M. L. (2008). Identification of a Highly Specific Hydroxyapatite‐binding Peptide using Phage Display. Advanced Materials, 20(10), 1830-1836.