Part:BBa_K4879007

JcFATB Expression Construct

The transcriptional unit coding for JcFATB, designed for expression in Y. lipolytica.

The expression cassette was assembled together along with the JcFATA and the CvFAP expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.

Characterization

Cloning: The assembled gene(s) were then inserted into the pYLEX plasmid (sourced from the Yarrowia Lipolytica Expression Kit, procured from Yeastern Biotech), by the means of NEBuilder HiFi DNA Assembly. The expected clones were then digested with NotI and SalI. The resulting digestion product is run on a 0.8% agarose gel. The cloning was confirmed, with the cloning success also being verified with sequencing. The linearized fragment was expected to be at above the 10 kbp band in the ladder (~13,000 bp)

Fig: Lanes 1-4 contain the possible clones and lane 5 contains the ladder. Clones in lanes 1,3, and 4 are likely successful clones.

Expression: To verify the expression of JcFATA, the coding sequence had a FLAG tag attached to the C-terminus. Subsequently, a Western blot was done for the protein after normalization.

The desired bands were visible in the blot but in the control too, as a result of possible contamination in the control. Thus, further experiments are needed to troubleshoot the problem and characterize the part fully. Depending on the refined results of the experiments, the functional validity of the part will be confirmed for future use.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 1803
  Illegal suffix found in sequence at 2073
  Illegal EcoRI site found at 1368
  Illegal EcoRI site found at 2067
  Illegal PstI site found at 587
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1368
  Illegal EcoRI site found at 1803
  Illegal EcoRI site found at 2067
  Illegal SpeI site found at 2074
  Illegal PstI site found at 587
  Illegal PstI site found at 2088
  Illegal NotI site found at 1809
  Illegal NotI site found at 2081
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1368
  Illegal EcoRI site found at 1803
  Illegal EcoRI site found at 2067
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 1803
  Illegal suffix found in sequence at 2074
  Illegal EcoRI site found at 1368
  Illegal EcoRI site found at 2067
  Illegal PstI site found at 587
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 1803
  Illegal EcoRI site found at 1368
  Illegal EcoRI site found at 2067
  Illegal XbaI site found at 1818
  Illegal SpeI site found at 2074
  Illegal PstI site found at 587
  Illegal PstI site found at 2088
  
• 1000
  COMPATIBLE WITH RFC[1000]