Part:BBa_K4879009

Yarrowia lipolytica HygR construct

The transcriptional unit for HygR, designed for expression in Y. lipolytica in order to delete the faa1 gene from our chassis.

Characterization

Cloning: The construct, along with homologous flanking sequences corresponding to the faa1 gene locus, were synthesized by iGEM’s DNA synthesis partner IDT. The resulting gene fragment was then PCR amplified with the respective primers to attain a higher copy number of the fragment. The amplification of the fragment was verified by running a 0.8% agarose gel, with the fragments expected to show at around the 3000 bp region.

Fig: Lane 1 is the DNA ladder, and lanes 4-7 contain the successfully amplified fragments

The amplified fragment was then gel-extracted and subsequently transformed into our chassis.

Validation: The transformed yeast culture was then plated onto the selection plates containing about 200 µg/ml of hygromycin B and were incubated at 30°C. The growth of the yeast on the selection plates confirms the functional validity of the expression construct.

Fig 2: The growth of transformed Y. lipolytica on the hygromycin B selection plate, confirming part functionality.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1561
  Illegal suffix found in sequence at 1831
  Illegal EcoRI site found at 684
  Illegal EcoRI site found at 778
  Illegal EcoRI site found at 1825
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 684
  Illegal EcoRI site found at 778
  Illegal EcoRI site found at 1561
  Illegal EcoRI site found at 1825
  Illegal SpeI site found at 1832
  Illegal PstI site found at 1846
  Illegal NotI site found at 1567
  Illegal NotI site found at 1839
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 684
  Illegal EcoRI site found at 778
  Illegal EcoRI site found at 1561
  Illegal EcoRI site found at 1825
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1561
  Illegal suffix found in sequence at 1832
  Illegal EcoRI site found at 684
  Illegal EcoRI site found at 778
  Illegal EcoRI site found at 1825
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1561
  Illegal EcoRI site found at 684
  Illegal EcoRI site found at 778
  Illegal EcoRI site found at 1825
  Illegal XbaI site found at 1576
  Illegal SpeI site found at 1832
  Illegal PstI site found at 1846
  
• 1000
  COMPATIBLE WITH RFC[1000]