Part:BBa_K192000:Design

Encapsulin

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 74
  Illegal XhoI site found at 274
  Illegal XhoI site found at 568
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

Sequence was PCR'd from T. Maritima. An additional tga stop site was added.

Helpful Tips

TD-PCR

• Forward Primer
  • 5'CTT CTA GAT GGA ATT TCT GAA AAG ATC

• Backward Primer
  • 5'CTA CTA GTA TCA TCA GAA CTT TAG AAG AAT CA

• TD-PCR amplification temperatures:

Notes

• Primers
  • The primers used and noted above contain a SpeI site downstream and a Xbal site upstream for "easy" ligation into a biobrick plasmid.
• Polymerase that was used was KapaHiFi (Engineered variant of of Taq)
  • Required to increase the [] of MgCl2.
  • After amplification at the specified TD-PCR program there was no unexpected bands

Suggestions

• Primers
  • For better enzymatic digestion of SpeI and Xbal it was suggested to extend the primers beyond the recognition sequence by a few base pairs to allow something for the enzymes get a "grip-on"
    • As long as 3' end of primer is complementary there should be no issues with primer annealing.
  • TD-PCR was done to ensure we had product to extract and subject to another round of PCR to optimize , however due to lack of non-specific products the PCR protocol was never optimized.

Source

Thermotoga Maritima