Part:BBa_K1329001

Hag-KpnI

The designed sequence was synthesized by Integrated DNA Technologies.

Application

As the original flagellin gene (Hag) contains multiple forbidden iGEM restriction sites the complete sequence of Hag-KpnI was synthesized by Integrated DNA Technologies. We inserted the KpnI restriction site because it does not occur in the iGEM prefix and suffixes of the shipping vector pSB1C3. This Brick was designed as a platform for the insertion of additional domains via Gibson assembly after linearizing the iGEM backbone pSB1C3-Hag-KpnI. The modified flagellin can afterwards be implemented into Bacillus subtilis.

Examples:

• Cup1-1
• D2-Strep

Test restriction with EcoRI and PstI:

The complete constructs were created by Gibson assembly. 5 clones of both Gibson assembly reactions have been tested for the insertion of the fragment.

According to the shift visible for 3 of the 5 Cup1 clones, these 3 clones can be considered as positive. Besides the 5 D2-Strep clones are presumably positive since the D2-domain contains an additional PstI restriction site that has to be removed later on.

An additional PCR has been performed with the isolated plasmids and primers that bind in the inserted domain and flagellin suffix. The PCR product further confirms that the domains have been inserted.

In the end the constructs have been sequenced which finally confirmed that hag-KpnI can be used as a platform for the insertion of multiple different domains into the flagellin of B. subtilis.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 588