Difference between revisions of "Part:BBa K4879007"

 
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The transcriptional unit coding for JcFATB, designed for expression in <i>Y. lipolytica</i>.
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The expression cassette was assembled together along with the JcFATB and the CvFAP expression cassettes with the help of the NEBuilder HiFi DNA Assembly kit.
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===Characterization===
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<b>Cloning:</b>
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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)
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<html><img src="https://static.igem.wiki/teams/4879/wiki/bba-k4879000-gel.jpg"width="720"height="720"></html>
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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.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 15:58, 11 October 2023


JcFATB Expression Construct

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

The expression cassette was assembled together along with the JcFATB 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.


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]