Difference between revisions of "Part:BBa K4879006"

 
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The transcriptional unit coding for JcFATA, 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 15:57, 11 October 2023


JcFATA expression construct

The transcriptional unit coding for JcFATA, 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 1642
    Illegal suffix found in sequence at 1912
    Illegal EcoRI site found at 1906
    Illegal PstI site found at 862
    Illegal PstI site found at 1469
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1642
    Illegal EcoRI site found at 1906
    Illegal SpeI site found at 1913
    Illegal PstI site found at 862
    Illegal PstI site found at 1469
    Illegal PstI site found at 1927
    Illegal NotI site found at 1648
    Illegal NotI site found at 1920
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1642
    Illegal EcoRI site found at 1906
    Illegal BglII site found at 1311
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1642
    Illegal suffix found in sequence at 1913
    Illegal EcoRI site found at 1906
    Illegal PstI site found at 862
    Illegal PstI site found at 1469
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1642
    Illegal EcoRI site found at 1906
    Illegal XbaI site found at 1657
    Illegal SpeI site found at 1913
    Illegal PstI site found at 862
    Illegal PstI site found at 1469
    Illegal PstI site found at 1927
  • 1000
    COMPATIBLE WITH RFC[1000]