Part:BBa_K4734005:Design
RBHS iGEM Designed Epitope for T. Gondii
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We designed the epitope ourself using software modeling. We then had to insert overhands on the sequence in order for the epitope to insert directly into the phage.
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.
Source
We got this epitope from the article listed above and it had the great level of antigenicity. The overhangs were added onto the epitope sequence on both ends in order for it to directly insert itself into the phage.