Translational_Unit

Part:BBa_K4879010

Designed by: Apoorva Gopal   Group: iGEM23_IISER-Pune-India   (2023-10-08)


Yarrowia lipolytica guaB construct

The transcriptional unit for guaB, designed for expression in Y. lipolytica in order to help select for ScRAD52 transformants.

Characterization

Cloning: The construct, along with homologous flanking sequences corresponding to the A08 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, and lanes 7-10 have the successful amplicons.


The amplified fragment was then gel-extracted and assembled together with the ScRAD52 expression construct (BBa_K4879011) 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 2000 µg/ml of mycophenolic acid and was 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 1831
    Illegal suffix found in sequence at 2101
    Illegal EcoRI site found at 928
    Illegal EcoRI site found at 1382
    Illegal EcoRI site found at 2095
    Illegal SpeI site found at 192
    Illegal PstI site found at 157
    Illegal PstI site found at 1241
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 928
    Illegal EcoRI site found at 1382
    Illegal EcoRI site found at 1831
    Illegal EcoRI site found at 2095
    Illegal SpeI site found at 192
    Illegal SpeI site found at 2102
    Illegal PstI site found at 157
    Illegal PstI site found at 1241
    Illegal PstI site found at 2116
    Illegal NotI site found at 1837
    Illegal NotI site found at 2109
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 928
    Illegal EcoRI site found at 1382
    Illegal EcoRI site found at 1831
    Illegal EcoRI site found at 2095
    Illegal BamHI site found at 1444
    Illegal XhoI site found at 1214
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1831
    Illegal suffix found in sequence at 2102
    Illegal EcoRI site found at 928
    Illegal EcoRI site found at 1382
    Illegal EcoRI site found at 2095
    Illegal SpeI site found at 192
    Illegal PstI site found at 157
    Illegal PstI site found at 1241
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1831
    Illegal EcoRI site found at 928
    Illegal EcoRI site found at 1382
    Illegal EcoRI site found at 2095
    Illegal XbaI site found at 1846
    Illegal SpeI site found at 192
    Illegal SpeI site found at 2102
    Illegal PstI site found at 157
    Illegal PstI site found at 1241
    Illegal PstI site found at 2116
    Illegal NgoMIV site found at 1231
    Illegal NgoMIV site found at 1456
  • 1000
    COMPATIBLE WITH RFC[1000]


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