Part:BBa_K4734003

TNNEDEQ Epitope

Usage and Biology

We chose our two other epitopes from the article “Toxoplasma gondii Tyrosine-Rich Oocyst Wall Protein: A Closer Look through an In Silico Prism” by Asghari et al., who discovered nine linear B-cell epitopes shared among three web servers (BCPREDS, ABCpred, and SVMTriP) with “high antigenicity score and good water solubility and without allergenic properties.” Of those, we selected the B-cell epitope TNNEDEQ, with an antigenicity score of 1.8297, no allergenicity, and good solubility. We also selected the B-cell epitope QGNDEHSSQ, with an antigenicity score of 1.7231, no allergenicity, and good solubility. Thus, we rationalize that these epitopes will be effective in our product. The epitope was then inserted onto the phage M13KE in order for the phage to penetrate the mucus layer and the epithelial cells in order to reach the lymph and produce an immune response.

The following protocol can be followed to insert the epitope on the phage:

Extend the annealed duplex as follows (mix in the given order) H2O: 119 µl 10X NEBuffer 2: 20μl annealed duplex: 50μl 10 mM dNTP's: 8μl Klenow fragment (NEB #M0210 ) (5 Units/µl ): 3μl Total: 200 µl Incubate at 37°C for 10 minutes, then 65°C for 15 minutes. Save 4 µl for later analysis (Step 5).

The extended duplex is then cut using EagI and either, KpnI or Acc65I. We favor digestion as follows: Extension reaction: 196 µl H2O: 158 μl 10X NEBuffer 2: 40 μl EagI-HF (NEB #R3505 ) (10 Units/µl ): 5 μl KpnI (NEB #R0142 ) (10 Units/µl ): 6 μl Total: 400 µl

Extension reaction: 196 µl H2O: 154 μl 10X NEBuffer 4: 40 μl EagI-HF (NEB #R3505 ) (10 Units/µl ): 5 μl KpnI-HF (NEB #R3142 ) (10 Units/µl ): 5 μl Total: 400 µl Incubate at 37°C for 3–5 hours.* Purify the DNA by phenol/chloroform extraction, chloroform extraction and ethanol precipitation.

• EagI-HF is not recommended for > 1 hour digestions.

The ligated phage can then be transformed in F+ bacteria.

Sequence and Features

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]