Part:BBa_K4879011

ScRAD52 Expression Construct

The transcriptional unit for ScRAD52, designed for expression in Y. lipolytica in order to increase homologous recombination probability to about 95%.

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 1: Lane 1 is the DNA ladder, with lanes 8-11 showing successful amplicons.

The amplified fragment was then gel-extracted and assembled together with the guaB expression construct with the help of the NEBuilder HiFi DNA Assembly kit, and subsequently transformed into our chassis.

Validation: The transformed yeast culture was then plated onto the selection plates containing about 1000-2000 µg/ml of mycophenolic acid 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 MPA selection plate, confirming part functionality.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1780
  Illegal suffix found in sequence at 2050
  Illegal EcoRI site found at 2044
  Illegal SpeI site found at 192
  Illegal PstI site found at 157
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1780
  Illegal EcoRI site found at 2044
  Illegal SpeI site found at 192
  Illegal SpeI site found at 2051
  Illegal PstI site found at 157
  Illegal PstI site found at 2065
  Illegal NotI site found at 1786
  Illegal NotI site found at 2058
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1780
  Illegal EcoRI site found at 2044
  Illegal BglII site found at 766
  Illegal BglII site found at 896
  Illegal XhoI site found at 1586
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1780
  Illegal suffix found in sequence at 2051
  Illegal EcoRI site found at 2044
  Illegal SpeI site found at 192
  Illegal PstI site found at 157
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1780
  Illegal EcoRI site found at 2044
  Illegal XbaI site found at 1795
  Illegal SpeI site found at 192
  Illegal SpeI site found at 2051
  Illegal PstI site found at 157
  Illegal PstI site found at 2065
  Illegal NgoMIV site found at 1570
  
• 1000
  COMPATIBLE WITH RFC[1000]